Detection, Capture and Quantification of Biological Moieties from Unprocessed Bodily Fluids Using Nanoplasmonic Platform

ABSTRACT

A nanoplasmonic platform can be used for the detection and quantification of multiple HIV subtypes in whole blood with localized surface plasmon resonance. Among other things, this nanoplasmonic platform provides a viable way to detection and quantification of viral load at a point of care with significantly less cost, time, and laboratory resources than existing methods of detection. Although an example of HIV detection in whole blood is provided, the nanoplasmonic platform is adaptable to detect other pathogens and infectious agents or macromolecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/668,274 entitled “Nanodetection of Viral Load fromWhole Blood” filed on Jul. 5, 2012 and claims the benefit of U.S.Provisional Patent Application No. 61/781,399 entitled “Nanodetection ofPathogen Load from Whole Blood” filed on Mar. 14, 2013. The contents ofthese applications are incorporated by reference herein in theirentirety for all purposes.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with government support under R01 A1081534, R01EB015776, R21 A10871, R01 AI093282, U54 EB15408, and R21 AI087107awarded by the National Institutes of Health and 106325 awarded by theDepartment of Defense. The United States government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

This invention relates to the detection of biological moieties such asinfectious agents, pathogens, or biomarkers. In particular, thisinvention relates to a nanoplasmonic platform for the detection andquantification of one or more pathogens such as HIV, HBV, or E. coli ata point-of-care (POC) using localized surface plasmon resonance (LSPR)principle.

Detection of infectious agents or pathogens (e.g., virus, bacteria, andfungi) is critical for homeland security, public and military health. Inrecent years, infectious diseases, especially viral outbreaks such asSARS, H1N1 and HIV, have a tremendous global healthcare impact, sincesuch viruses could rapidly evolve, spread, and turn into pandemics suchas HIV/AIDS and Spanish flu that had a devastating global impact causingtotally over 80 million deaths. To identify and control forthcomingepidemics, it will be a critical to develop rapid, reliable, accurate,and sensitive diagnostic technologies that have the ability to betailored to multiple settings.

Effective, rapid, accurate, and simple detection of complex pathogensand infectious agents still poses significant biological and engineeringchallenges. Among other things, biological challenges can arise due tothe presence of multiple subtypes and strains of the pathogen that makesit difficult to achieve repeatable and reliable capture efficienciesfrom bodily samples without demanding lengthy sample preparation steps.

To provide one exemplary case of the cost of this unaddressed detectionneed, it is estimated that annually over 450,000 infants are infectedwith HIV through mother-to-child transmission (MTCT), which is theprimary cause of AIDS in children. Rapid progression of AIDS in infantscauses early death. Combined data from nine clinical trials in Africashowed that 35% of HIV-1 positive infants die by the age of 1 and 52% ofHIV-1 infected children die by the age of 2.

Studies have shown that long-term suppression of HIV-1 replication ininfants can be achieved by initiating antiretroviral therapy (ART) toreduce AIDS-related morbidity and mortality, and improve the quality oflife. In order to provide early AIDS care and ART to HIV-infectedinfants, early diagnosis is key as ART is not provided until infectionhas been established.

However, simple and rapid serological assays cannot detect HIV-infectedinfants until 18 months after birth. The late identification of AIDSchildren is due to the interference of maternal HIV-1 specificantibodies, which are passively transferred through the placenta andpersist in infants for approximately 18 months. Unfortunately, the viralload assays used in developed countries are expensive and requiresignificant instrumentation. Thus, the lack of cost-effective POC viralload assays that can effectively reach patients living in rural,isolated settings prevents identifying HIV-infected infants that wouldgreatly benefit from starting ART. Thus, the monitoring the viral loadof HIV at a POC for many patients, and infants in particular, is stillan unaddressed challenge. Traditional detection techniques such asculturing, enzyme-linked immunosorbent assay (ELISA) and polymerasechain reaction (PCR) cannot be implemented at POC settings withoutequipped laboratories and extensive infrastructure.

In developed countries, HIV-1 viral load is monitored using commercialRNA assays, such as Roche COBAS®, Abbott RealTime, Siemens Versant™ andbioMerieux NucliSens®. However, implementation of these assays requiresexpensive equipment (e.g., thermal cyclers, $20,000), highly skilledpersonnel, and expensive reagents ($50-200 per test in the US), which inresource-constrained settings is unaffordable and unsustainable.Alternative viral load assays have been developed, such as theUltrasensitive p24 assay, the ExaVir™ RT viral load assay, and real-timereverse transcriptase quantitative polymerase chain reaction (RT-qPCR).However, these assays are still costly requiring refrigeration andskilled operators. Additionally, throughput of the ExaVir™ RT viral loadassay is low, with turnaround time of two days and is limited to 180samples per week per operator. Miniaturized conventional ELISA fordetection of p24 antigen or RT-qPCR for detection of HIV-1 RNA has beendeveloped. However, these methods require complex on-chip designs due tomulti-step manipulations such as labor-intensive sample preparation(plasma separation and RNA extraction), amplification (expensivereagents) and detection. Additionally, these methods require paralleltesting of external standards for a standard curve, which furtherincrease the device complexity. Thus, current viral load assays, due totechnical requirements and costs, are not available to benefit AIDSpatients at the POC in resource-constrained settings.

For early identification of HIV-positive infants, isolation of HIV-1 incell cultures was initially used, although it was time-consuming, costlyand technically demanding. Most commonly, MTCT is diagnosed via PCR toamplify HIV-1 DNA integrated into the genome of white blood cells(WBCs). However, performing PCR still requires highly-trained operators,is time-consuming, and more expensive than current rapid serologicalassays. There have been efforts to develop miniaturized PCR chipsincluding chips employing RT-qPCR for HIV-1 RNA detection.

Thus, there remains a generalized need for the detection andquantification of one or more infectious agents or pathogens at the POC.More specifically, there is a need for new, simple, highly sensitive,specific, accurate, reliable, rapid, and feasible viral load assays thatare necessary to avoid further infectious agent and pathogen propagationand to screen for initiating early treatment at the inception of anepidemic.

SUMMARY OF THE INVENTION

Infectious diseases such as SARS and HIV pose an omnipresent danger toglobal health. Reliable, fast, accurate and sensitive platforms that canbe deployed at the point-of-care (POC) in multiple settings, such asairports and offices for detection of infectious pathogens are essentialto interfere in epidemics and possible biological attacks.

Disclosed herein is a broadly applicable technology for quantitative,nanoplasmonic-based intact multiple pathogen and infectious agentdetection at clinically relevant concentrations without any samplepreparation directly from whole blood. The disclosed sensing platform isbased on the unique nanoplasmonic properties of nanoparticles and theimmobilization of antibodies for rapidly evolving subtypes of viruses.

Although this technology will be described with reference to viral loaddetection of various subtypes of HIV below, this technology may beequally applicable and adaptable to the detection of other biologicalmoieties including, but not limited to, infectious agents, pathogens,organisms, or reaction products (including peptides, nucleic acids,peptide nucleic acids, carbohydrates, and the other biological andchemical relevant products) by changing the surface chemistry forpathogens, organisms, or reaction products having reasonablywell-described markers available. For example, instead of viraldetection the technology might be used to detect other pathogens,infectious agents, organisms, or reaction products such as bacteria,virus, fungi, nucleic acids, peptides, secreted cellular products, cellfragments, exosomes, and so forth. For example, E. coli may be detected.This technology is also broadly applicable to other types of infectiousdiseases having reasonably well described biomarkers availableincluding, but not limited to, influenza, hepatitis, malaria, dengue,epilepsy, and tuberculosis (tuberculosis might be found in sputum).Other (bio)markers such as KIM-1 (kidney injury molecule-1), which is abiomarker for human renal proximal tubule injury, might also be detectedusing this platform as just one example. This technology may also beutilized in the detection of bacteria, related biological macromoleculesof bacteria, and neutrophils in peritoneal dialysis bags. Moreover, thistechnology may be applicable to detection of the products of reactionsincluding, but not limited to, polymer reactions such as cross-linkingand synthesis reactions, enzymatic reactions, and hydrolysis reactions.In some instances, a phase change may be detectable and/or a change insize or molecular weight. Additionally, while the samples provided inthe examples below are blood samples, other types of samples or bodilyfluids may be used including, but not limited to, serum, urine, saliva,vaginal secretions, and sputum. These samples may be obtained fromhumans or from animals and may contain mammalian cells; moreover, thesesamples may include plant cells, yeast and fungi.

In this disclosure, the capture of multiple HIV subtypes (A, B, C, D, E,G, and subtype panel) on a biosensing surface of a nanoplasmonicplatform is presented as well as the detection and quantification of thevirus using localized surface plasmon resonance principle. These resultswere compared to RT-qPCR as a gold standard and the disclosed systempresented high repeatability (56-90%). Preliminary results have shownthat the nanoplasmonic platform results correlated significantly forviral load concentrations ranging from 50 to 10⁶ copies/mL in spikedwhole blood samples and HIV-infected patient blood samples, indicatingthat the microchip performs accurately with a clinically reasonableerror. This method offers an assay time of 1 hour and 10 minutes (1 hourfor capture, 10 minutes for detection and data analysis). This assaytime can be shortened by changing the volume of samples and optimizingthe biosensing platform dimensions. These samples corresponded toconcentrations that cover the treatment failure range including thecurrent definitions of the World Health Organization (WHO), Departmentof Health and Human Services (DHHS) and AIDS Clinical Trials Group(ACTG).

The disclosed system is shown to be accurate, repeatable, and reliableto capture intact viruses without any damage on the virus structure andcharacteristics including their capsid structure and genetic content.This capture can occur with high capture specificity and efficienciesusing immuno-surface chemistry approaches directly from whole bloodsamples without any sample preprocessing step.

This nanoplasmonic platform may be implemented in a lab on a low-costmicrofluidic chip that is subjected to LSPR on location to attain atimely and accurate pathogen and/or infectious agent count in wholeblood sample. In the examples below, the viral load microchips cancapture HIV-1 with high efficiency and specificity (which is confirmedby the examples below) and are readable with a portable lensless chargecoupled device (CCD)-based LSPR technology for label-free detection andquantification of HIV-1. The use of CCD system eliminates the need forfluorescence imaging. The combination of the microchip with the lenslesssystem provides a novel portable and battery-powered viral load systemfor resource-constrained settings. This overcomes many of thedeficiencies with existing technologies for viral load detection whichare limited by required equipment, time, cost, and other processingfactors.

Thus, this broadly applicable platform holds great promise to become adetection platform at the POC settings in rural and remote areas as wellas in the hospital and primary care settings. Further, these approacheshave wide implications and potential to be applicable in the U.S. aspersonalized bed-side diagnostic technologies. As an example, suchmicrochips can also potentially be used to capture, detect and rapidlyquantify other viruses such as dengue and H1N1 as well as oncovirusesand circulating tumor cells (CTCs are one per billion blood cells,peritoneal cavity fluid, and lymph circulation fluid) for prevention andtreatment.

These and still other advantages of the invention will be apparent fromthe detailed description and drawings. What follows is merely adescription of a preferred embodiment of the present invention. Toassess the full scope of the invention, the claims should be looked toas the preferred embodiment is not intended to be the only embodimentwithin the scope of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate an exemplary nanoplasmonic platform for viralload detection.

FIGS. 2A and 2B illustrate the surface modification of polystyrene (PS)surfaces using poly-l-lysine (PLL) and the spectral peak shifts andextinction intensities corresponding to the application of varying PLLconcentrations on the PS surface.

FIGS. 3A and 3D illustrate the observed wavelengths over alayer-by-layer surface preparation of the nanoplasmonic platform and theobserved wavelength peak shift between a control whole blood sample andvarious whole blood samples spiked with a subtype or subtype panel ofHIV. FIGS. 3B and 3C illustrate the repeatability parameter for surfacechemistry in terms of wavelength and extinction intensity, respectively.

FIGS. 4A through 4H illustrate the original wavelength spectra and curvefitting analysis for multiple HIV subtypes.

FIGS. 5A through 5D provide surface characterization of PS surfacesbefore and after surface modification steps using Atomic ForceMicroscopy (AFM); FIG. 5E includes two scanning electron microscopeimages of the captured intact viruses on the antibody immobilizedbiosensing surface.

FIGS. 6A through 6H provide validation of the nanoplasmonic platformwith spiked in whole blood using curve fitting method and HIV-infectedpatient samples using RT-qPCR by comparing a detected change inwavelength to the viral load detected by RT-qPCR. FIG. 6I illustratesthe repeatability parameter for HIV spiked in whole blood andHIV-infected patient samples in terms of wavelength change.

FIGS. 7A through 7H provide validation of the nanoplasmonic platformwith spiked in whole blood using experimental data maximum method andHIV-infected patient samples using RT-qPCR by comparing a detectedchange in wavelength to the viral load detected by RT-qPCR. FIG. 7Iillustrates the repeatability parameter for HIV spiked in whole bloodand HIV-infected patient samples in terms of wavelength change.

FIGS. 8A through 8H provide validation of the nanoplasmonic platformwith spiked in whole blood and HIV-infected patient samples usingRT-qPCR by comparing a detected change in extinction intensity to theviral load detected by RT-qPCR. FIG. 8I illustrates the repeatabilityparameter for HIV spiked in whole blood and HIV-infected patient samplesin terms of extinction intensity change.

FIG. 9 provides further validation of the nanoplasmonic platform bycomparing the detected change in wavelength of a HIV subtype panelsuspension in phosphate buffer saline (PBS) with viral loads detectedusing RT-qPCR.

FIGS. 10A and 10B provide quantitative detection with HIV-infectedpatient samples and provide a comparison between the counts obtained bythe nanoplasmonic platform and RT-qPCR. In FIG. 10A, eight discardedHIV-infected patient samples in whole blood were evaluated usingnanoplasmonic platform, and the platform presented a viral load rangingfrom (1.3±0.7) log₁₀ copies/mL to (4.3±1.2) log₁₀ copies/mL. RT-qPCRcount presented a viral load ranging from (2.7±0.1) log₁₀ copies/mL to(3.6±0.1) log₁₀ copies/mL in patient samples. (n=5-6, error barsrepresent standard error of the mean (SEM)). FIG. 10B illustrates thatBland-Altman Analysis between the nanoplasmonic platform and RT-qPCRcounts did not demonstrate an evidence for a systematic bias for HIVviral load for HIV-infected patient blood samples.

FIG. 11 shows the steps for measuring a viral load on a microchip.

FIGS. 12A through 12C show the fabrication of a viral load microchip.FIG. 12A shows a schematic of a viral load microchip fabricationprocess. FIG. 12B shows a prototype viral load microchip. FIG. 12C showsa top view schematic of a disposable microchip and fluid reservoirs inwhich the chip has fluidic interface between microchannels.

FIG. 13 illustrates the observed wavelengths over a layer-by-layersurface preparation of a flexible nanoplasmonic platform.

FIG. 14 illustrates the different methods used for antiepileptic drugquantification. The gold standard method required expensive HPLCequipment and skilled technician and is shown as the upper path that islab based. In contrast, localized surface plasmon resonance baseddetection can be applied in point-of-care settings as is illustrated inthe lower path.

FIG. 15 illustrates the design of a microfluidic LSPR microchip for AEDquantification. In FIG. 15A(i), the surface is functionalized with goldnanoparticles and anti-Carbamazepine (CBZ) antibody for specificdetection of CBZ molecules. In FIG. 15A(ii), a droplet of blood fromfingerprick is injected into microfluidic channels. Then, the extinctionwavelength shift is measured using a portable spectrophotometer, theresults of which are generally shown in FIG. 15B in which wavelengthshift due to the specific binding of CBZ molecules to the anti-CBZantibody is illustrated.

FIG. 16 illustrates another evaluation of the nanoplasmonic platformtechnology with HIV samples. FIG. 16A provides the aggregated data forthe validation of the nanoplasmonic platform with multiple HIV subtype(A, B, C, D, E, G) spiked in whole blood and HIV-infected patientsamples from FIG. 6A-6H in a single chart. The limit-of-detection wascalculated for each subtype, and compared to the control sample. (i.e.,whole blood without HIV) as is illustrated in FIG. 16B. The presentedwavelength shifts of multiple HIV subtype samples are significantlydifferent than the control sample (Statistical assessment on the resultswas performed using non-parametric Kruskal-Wallis one-way analysis ofvariance followed by Mann-Whitney U test with Bonferroni correction formultiple comparisons. Statistical significance threshold was set at0.05, p<0.05).

FIG. 17 is a top view schematic of a disposable cartridge of a fluidicmicrochip and fluid reservoirs. The chip has fluidic interface betweenthe microfluidic channels.

FIG. 18 illustrates the original wavelength spectra and demonstrates thewavelength shift after the application E. coli.

FIG. 19A provides validation of the nanoplasmonic platform with spikedin PBS using curve fitting method in terms of wavelength change. FIG.19B illustrates the repeatability parameter for E. coli spiked in PBSsamples in terms of wavelength change.

FIG. 20A provides validation of the nanoplasmonic platform with IFN-γspiked in PBS samples using curve fitting method in terms of wavelengthchange. FIG. 20B illustrates the repeatability parameter for IFN-γspiked in PBS samples in terms of wavelength change.

FIG. 21A provides validation of the nanoplasmonic platform for HBV-HBcdetection using curve fitting method in terms of wavelength change. FIG.21B illustrates the repeatability parameter for HBV-HBc spiked samplesin terms of wavelength change.

FIG. 22A provides validation of the nanoplasmonic platform for HBV-HBsdetection using curve fitting method in terms of wavelength change. FIG.22B illustrates the repeatability parameter for HBV-HBs spiked samplesin terms of wavelength change.

DETAILED DESCRIPTION

Biosensing platforms are disclosed herein for direct intact multiplepathogen and/or infectious agent detection using the nanoplasmonicproperties of nanoparticles. These disclosed technology platforms andsystems take a significant step toward providing POC tests atresource-constrained settings as well as hospital and primary caresettings.

It should be noted that label-free biodetection platforms such aselectrical, mechanical and optical mechanisms have been recently usedfor the detection and diagnostics of infectious agents. These biosensingtechnologies offer multiple pathogen and disease detection applicationsranging from laboratory research to medical diagnostics and drugdevelopment/treatment to engaging biologic threats.

A nanoplasmonic platform for the detection and quantification of one ormore pathogens in a bodily fluid sample, such as a whole blood sample,is disclosed. The nanoplasmonic platform includes a substrate having animmobilization layer, a plurality of gold nanoparticles immobilized onthe immobilization layer, and one or more antibodies linked to the goldnanoparticles in which the antibodies are configured to selectively bindto the pathogen(s) and infectious agent(s). The nanoplasmonic platformis adapted for the detection and quantification of the pathogen(s) andinfectious agent(s) using localized surface plasmon resonance (LSPR).This technology may be broadly applicable to the detection of any typeof biological moiety that has a corresponding recognition element (e.g.,antibody), and it is contemplated that nanoparticles other that goldnanoparticles might be employed. As some non-limiting examples, therecognition elements include one or more of an anti-gp120 antibody, ananti-gp41 antibody, an anti-gp24 antibody, and lectin and therecognition elements could be adapted to detect E. coli or HBV.

When describing the nanoplasmonic platform and binding of the antibodythereto, terms such as linked, bound, connect, attach, interact, and soforth should be understood as referring to linkages that result in thejoining of the elements being referred to, whether such joining ispermanent or potentially reversible. These terms should not be read asrequiring the formation of covalent bonds, although covalent-type bondmight be formed.

The substrate of the nanoplasmonic platform can be optically transparentto facilitate LSPR. Polystyrene, glass parylene, quartz crystal,graphene and mica layers, and poly(methyl methacrylate) are goodcandidates for the substrate. They are good candidates because they areoptically transparent and are capable of supporting the functionalizedgold nanoparticles which selectively bind to the pathogen(s) andinfectious agent(s) via the surface recognition elements such asantibodies and which possess the nanoplasmonic properties thatfacilitate LSPR detection of the binding detection and capture events.

The immobilization layer of the nanoplasmonic platform can be, forexample poly-l-lysine (although other layers may be able to immobilizethe nanoparticles on the substrate and not interfere with LSPRdetection), and can have amine-terminated groups that are used toimmobilize the plurality of gold nanoparticles. The immobilization layermay be functionalized with at least one of the metal binding groups suchas amine groups or thiol groups, although other attachments between thelayer and the substrate or the gold nanoparticles may also be employedas long as it does not impair the ability of the nanoplasmonic platformto be read using LSPR.

To link the one or more surface recognition elements such as antibodiesto the gold nanoparticles, a modified support surface may be formed bypreparing a surface of the plurality of gold nanoparticles using amercaptoundecanoic acid to form carboxyl groups, reactingN-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride with thecarboxyl groups to form an amine reactive intermediate, and stabilizingthe amine reactive intermediate by the of N-hydroxysulfosuccinimide toform the modified support surface. The modified support surface may belinked to a NeutrAvidin protein which is used to immobilize ananti-gp120 antibody. The anti-HIV gp120 antibody accommodates thedetection of various subtypes of HIV because this antibody is adapted tobind to multiple subtypes of HIV. Additionally, other or additionalantibodies may be present based on the pathogens and infectious agentsto be detected. It is contemplated that multiple pathogens may bedetected on a single nanoplasmonic platform. In some embodiments, theantibody may be a polyclonal antibody. In the context of HIV detection,the antibody may be, for example, a gp120 antibody, a gp41 antibody, agp24 antibody, or lectin, all of which are able to detect multiplesubtypes of HIV. Additionally, in some forms, the modified supportsurface is linked to at least one of a protein A, a protein G, a proteinA/G, a Streptavidin protein, and a NeutrAvidin protein which is used toform chemical bonds to immobilize recognition elements such as theantibody on the modified support surface.

After the binding of an antigen to the antibody, the nanoplasmonicplatform exhibits an observable change in wavelength, in at least one ofwavelength shift and extinction intensity, when subjected to LSPR incomparison to when no binding has occurred due to the exceptionalnanoplasmonic properties of the gold nanoparticles.

The nanoplasmonic platform may be based on a microfluidic device havingan inlet for reception of the sample in which the inlet is in fluidcommunication with a capture detection channel that includes one or moreantibodies linked to the plurality of gold nanoparticles therein. Themicrofluidic device can further comprises a filter disposed between theinlet and the capture detection channel in which the filter has aporosity that filters the sample to produce a filtered sample forselectively binding to the at least one antibody. In this way, thecapture efficiency of the system can be improved.

A method of making a nanoplasmonic platform of the type described aboveis also disclosed. This method includes depositing an immobilizationlayer on a substrate (which in some forms may be poly-l-lysine),immobilizing a plurality of gold nanoparticles on the immobilizationlayer, and linking one or more antibodies to the gold nanoparticles inwhich the one or more antibodies are configured to selectively bind topathogen(s) and infectious agent(s).

As mentioned above, the substrate may be optically transparent tofacilitate LSPR measurements and may include polystyrene, glass,parylene, quartz crystal, graphene and mica layers, and/or poly(methylmethacrylate).

Again, the antibody attached to the gold nanoparticles may be ananti-HIV gp120 antibody and pathogen and infectious agent may includeHIV. Alternatively, polyclonal antibodies may be used as well as any ofthe antibodies describe above may be used to detect HIV. Again, some ofthe antibodies that may be used to detect HIV include gp120, gp41, gp24,and lectin.

According to one specific form of the method, a modified support surfacefor linking the at least one antibody to the gold nanoparticles may beformed by the steps of preparing a surface of the plurality of goldnanoparticles using a mercaptoundecanoic acid to form carboxyl groups,reacting N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloridewith the carboxyl groups to form an amine reactive intermediate, andstabilizing the amine reactive intermediate using the ofN-hydroxysulfosuccinimide to form the modified support surface. Themodified support surface may be linked to a NeutrAvidin protein which isused to immobilize an anti-HIV gp120 antibody.

Also disclosed herein is a method of detecting a pathogen and/orinfectious agent in a sample using the nanoplasmonic platform. Themethod of detection includes receiving the sample on the nanoplasmonicplatform to capture a pathogen and/or infectious agent and performinglocalized surface plasmon resonance on the nanoplasmonic platform toobtain a wavelength signal and extinction intensity corresponding to thesample received on the nanoplasmonic platform.

The wavelength signal corresponding to the sample received on thenanoplasmonic platform may be used in a comparison to determine whetherthe pathogen has bound to the at least one recognition element such asan antibody on the nanoplasmonic platform. This permits for thedetection of a pathogen and/or infectious agent or pathogen subtype bythe nanoplasmonic platform as well as a quantity of the pathogen and/orinfectious agent in the sample (i.e., a viral load count in anunprocessed whole blood sample, a serum sample). The wavelength signalthat is read and used as a basis for comparison may be, for example, awavelength shift and/or an extinction intensity. The examples providedbelow relay experimental data when the pathogens being detected ismultiple subtypes of HIV.

The nanoplasmonic platform may be based on a microfluidic device. Themicrofluidic device may have an inlet for the reception of the sample inwhich the inlet is in fluid communication with a capture detectionchannel that includes at least one antibody linked to the plurality ofgold nanoparticles. The step of receiving the sample on thenanoplasmonic platform may include flowing the sample through thecapture detection channel to selectively bind the pathogen and/orinfectious agent to the at least one recognition element such as anantibody before the sample is subjected to localized surface plasmonresonance. In some forms, the microfluidic device can further include afilter disposed between the inlet and the capture detection channel andthe method further includes the step of filtering the sample to producea filtered sample prior to the step of selectively binding the pathogenand/or infectious agent to the at least one antibody in the capturedetection channel.

Below, we demonstrated for the first time HIV viral load quantificationusing a nanoplasmonic optical detection system using multiple subtypesin HIV-infected patient samples as a model system. Here, we present forthe first time a reliable, feasible, label-free, fluorescence-free andrepeatable technology that captures multiple viral subtypes fromunprocessed whole blood, and subsequently quantifies and reports viralload.

One exciting application of this technology is in infant testing whereHIV status cannot be monitored by white blood cell counting, and viralload assays are required. The presented nanoplasmonic platformtechnology detected virus particles in whole blood of HIV-infectedpatients, which contain HIV antibodies. Since antibodies in men andwomen would not be any different, antibodies transferred from women totheir infants would not be expected to have any greater effect ininterfering with detection (i.e., if antibodies do not interfere withdetection in infected adults, passively transferred antibodies will notinterfere with detection in infants). In the clinical samples tested sofar, no interference effects have been observed.

Specific examples of the process or nanoplasmonic platform, method ofmaking this nanoplasmonic, and method of detecting pathogens and/orinfectious agents are provided below. These examples are offered forillustrative purposes only, and are not intended to limit the scope ofthe present invention in any way. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andthe following example and fall within the scope of the appended claims.

Example I

Referring first to FIGS. 1A and 1B, a nanoplasmonic viral load detectionplatform is illustrated. FIG. 1A shows the capture of HIV on theantibody immobilized biosensing surface and FIG. 1B illustrates thestructure of the nanoplasmonic platform and various layers. Theconstruction of the exemplary nanoplasmonic platform is now describedalong with data characterizing of the platform, including someintermediate forms of the platform.

The nanoplasmonic platform is constructed using a polystyrene surface asa base substrate. For the prepared trials, polystyrene well plates werepurchased from Corning Inc. of Corning, N.Y. The polystyrene surfaceswere first washed with absolute ethanol and distilled water, then driedunder nitrogen gas. The ethanol (200 proof) was purchased from FisherScientific of Fair Lawn, N.J. Following cleaning steps, the polystyrenesurface was modified by poly-l-lysine (PLL) to form amine-terminatedgroups. A series of densities of PLL (from 0.01 to 0.1 mg/mL) in 1×phosphate buffer saline (PBS) were evaluated, and then, surfaces wereincubated at 4° C. overnight. The PLL was purchased from Sigma Co. ofSt. Louis, Mo. and the phosphate buffered saline (PBS, pH=7.4, 1×) waspurchased from Invitrogen Co. of Carlsbad, Calif.

After PLL modification, 40 μL of gold nanoparticle solution was loadedonto each surface and incubated at 4° C. overnight for binding andseeding of nanoparticles onto the support material. The goldnanoparticles, having a 10 nm diameter, were purchased from TedPella ofRedding, Calif.

To enable the capture of HIV on the biosensing surface, after thestarting polystyrene surface was modified by poly-l-lysine (PLL) togenerate amine-terminated groups and gold nanoparticles (AuNP) wereimmobilized on the amine-terminated surface, then additional layers wereprepared to support and immobilize the antibody of interest. The goldnanoparticle immobilized surface was first activated by application of100 μL of 1 mM 11-Mercaptoundecanoic acid (MUA) dissolved in ethanol.MUA forms carboxyl groups for crosslinking agents.N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (100mM EDC dissolved in 50 mM MES buffer at pH 5.0) reacts with the carboxylgroups to form amine reactive intermediate. EDC-mediated coupling wasstabilized by the addition of N-hydroxysulfosuccinimide (NHS) (50 mM NHSdissolved in 50 mM MES buffer at pH 5.0). After EDC/NHS couplingreaction, 100 μL of NeutrAvidin™ (NA) (0.1 mg/mL) was placed on themodified support surface to immobilize antibodies on the surface andenhance the capture efficiency. To minimize nonspecific binding on bothinactive and reactive areas of the surface, 100 μL of 10% BSA was usedas a blocking agent on each surface and incubated at 4° C. for an hour.After the BSA blocking step, 5 μg/mL of biotinylated anti-gp120polyclonal antibodies were placed onto biotin binding sites onNeutrAvidin by loading 100 μL of antibody solution onto each modifiedsurface and incubated at 4° C. for an hour. The MUA, EDC, NHS and BSA(10%) were obtained from Aldrich Chemical Co. of Milwaukee, Wis. MES waspurchased from Sigma Co. of St. Louis, Mo. NeutrAvidin™ protein wasobtained from Pierce Biotechnology of Rockford, Ill. Biotinylated, goatanti-HIV gp120 antibody was obtained from Abcam Inc. of Cambridge, Mass.

It is noted that metal nanoparticles have been used in drug delivery andclinical diagnostics, and their nanoplasmonic properties present theadvantage of light coupling upon the changes on the surface. Associationand/or dissociation onto metal nanoparticles lead to changes in theabsorbed wavelength in LSPR. Each modification on the metal nanoparticlesurface causes an extinction intensity change and, it enables a broadwindow for spectral measurements with a high signal-to-noise ratio andminimal background signal.

Thus, biosensing platforms incorporating nanoparticles have been used invarious biochemical sensing platforms and spectroscopies allowingpicomolar sensitivity to detect protein and nucleic acid interactions.Although localized surface plasmon resonance principles have beenutilized by others for protein and nucleic acid detection applications,intact viral detection and viral load quantification has not beenperformed from unprocessed whole blood.

However, there are a number of engineering and sampling challenges inboth production and clinical testing of metal nanoparticle-baseddetection methods. First, typical chemical and physical modifications ofnanoparticles (e.g., plasma treatment and exposure to highly acidicconditions) can lead to irreversible aggregation, resulting indegradation of optical characteristics. Also, existingnanoparticle-based pathogen detection methods have suffered from thechallenges associated with direct exposure to unprocessed whole blood,including non-specific binding and the requirement for extensive samplepreparation, thus reducing clinical relevance for POC applications.Currently, diagnostic tests require sample preprocessing includingplasma separation to prevent inaccuracy in signal amplification andquantification steps. On the other hand, it is a significant challengeto deploy these biosensing platforms into a multiplexed format that candetect multiple subtypes of pathogens from unidentified patient samples.

Example II

To evaluate gold nanoparticle binding and seeding on polystyrenesurface, a series of poly-l-lysine (PLL) concentrations (0.01 to 0.1mg/mL) were evaluated and a linear concentration-dependent wavelengthshift was observed as illustrated in FIG. 2A. The preparation of thesurfaces to evaluate gold nanoparticle incubation time onamine-terminated polystyrene surface was performed with 1-day and 3-dayincubation steps. FIG. 2A shows a statistically comparable correlationbetween wavelength shift and PLL concentration for 1-day and 3-dayincubation steps. Based on these results, it was established that theexperiments could be prepared using the 1-day incubation of PLL todecrease the incubation time for the preparation of the devices.

As shown in FIG. 2A, the individual wavelength of bare goldnanoparticles shifted from 518 nm to 546.4±1.6 nm, when PLLconcentration (1-day incubation) was increased from 0.01 to 0.1 mg/mL.The number of amine groups played a notable role in nanoparticle bindingonto the surface as it is believed that, at least up point, theconcentration of PLL increases the number of amine-terminated groups toimmobilize the gold nanoparticles. On the other hand, PLL concentrationsabove 0.1 mg/mL were observed to cause a collapsed polymer formation onthe substrate, and a further increase in the concentration of goldnanoparticle binding sites on the surface was not observed as isgenerally evidenced by FIG. 2B.

Wavelength peak shifts by gold nanoparticle immobilization at PLLconcentrations ranging from 0.01 to 0.1 mg/mL were then evaluated usinga 1-day PLL incubation step and are illustrated in FIG. 2B. In theexperiments, 0.05 mg/mL PLL concentration was chosen to avoid excessivedilution steps reducing possible variations in surface chemistry.Further, 0.05 mg/mL of PLL provided a high extinction coefficient atthis PLL concentration to maximize gold nanoparticle binding onto thePLL treated surface (n=6, error bars represent standard error of themean). Following each modification step, surfaces were rinsed with 1×PBSthree times to minimize possible variations in the wavelength andintensity measurements.

It should be noted that metal nanoparticles that vary in size, shape,and material present different maximum wavelength points. This propertyallows for tuning of the nanoplasmonic wavelength point throughout thevisible, near-infrared, and into the infrared region of theelectromagnetic spectrum, offering flexibility. To avoid batch-to-batchvariations and to create a reproducible platform, the various samplespresented herein used the same batch of gold nanoparticle solution forreproducible nanoplasmonic measurements.

Example III

At each surface modification and after an HIV capture step, spectralanalysis was monitored by either curve fitting analysis or experimentaldata maximum method, which reports the peak shifts at the maximumextinction wavelength. Each binding study was characterized with adetected shift of the maximum extinction point of gold nanoparticles byVarioskan® Flash Spectral Scanning Multimode Readers, Thermo Scientific.The measuring mode was set to scan the extinction changes per wavelengthfrom 400 nm to 700 nm over 301 steps. The detection light beam area ofthe spectrometer was indicated as 3.14 mm² (maximum). The spectralresolution and intensity accuracy of the instrument with fixed slitsetting was 1 nm and 0.003 a.u. extinction intensity. We performed 6replicates for nanoplasmonic measurements for all samples.

To analyze the wavelength data, we employed two approaches (i.e., curvefitting and experimental data maximum). In the first method, a softwarecode (e.g. MATLAB) was written to find the nanoplasmonic wavelength peakof each recorded spectra from curve fitting. A Fourier type expansionwith 8 harmonics (i.e.,

f(x)=a ₀+Σ_(n=1) ⁸(a _(n) cos(nωx)+b _(n) sin(nωx))

where ω is the fundamental frequency of the signal, a_(n) and b_(n) areexpansion coefficients) was used to fit to each recorded spectra. The R²values were found to be greater than 0.99 with the MATLAB fit command.The wavelengths at the maximum extinction value for each recordedspectra were extracted from the curve fits and were rounded to the firstdecimal digit considering the finite resolution of the experiment.Individual reference curve subtraction was performed for each HIVsubtype curve at a given virus concentration from the correspondingreference curve. All data for wavelength measurements were presented asthe mean of wavelength measurements±standard error of the mean (SEM).The experimental spectra evaluated by the experimental data maximummethod were compared with the Fourier type curve fitting analysis.

In the second data analysis method, nanoplasmonic wavelength peak wasdetermined as the wavelength at the maximum extinction value, and alldata for wavelength and extinction intensity measurements were presentedas the mean of wavelength or extinction intensity measurements±SEM. Foreach HIV subtype curve at a given virus concentration, individualreference curve subtraction was carried out from the correspondingreference curve. Considering the resolution of the instrument, the datawas presented with one decimal digit in the results, and the errors fromthe finite resolution of the spectrometer were considered in theanalysis.

Looking at FIG. 3A, each modification was analyzed using curve fittingmethod. To evaluate the surface chemistry, a control group of unmodified10 nm diameter gold nanoparticles were utilized. An individualwavelength peak of gold nanoparticles (AuNP) was repeatably observed at518 nm (n=6, p<0.05) as in FIG. 3A. Gold nanoparticle binding on 0.05mg/mL of the PLL-modified surface led to a statistically significantpeak shift from the gold coated surface from 518 nm to 546.7±1.8 nm(n=6, p<0.05).

As outlined above, following gold nanoparticle coating of the PLLmodified surface, MUA was self-assembled as a monolayer onto the goldnanoparticle layer and generated carboxyl groups on the surface of thegold nanoparticle layer.

Antibodies immobilized with a favorable orientation using NeutrAvidinhave a higher capture efficiency towards bioagents than thoseimmobilized by physical absorption and chemical binding of antibodiesonto a surface. To immobilize biotinylated antibodies for HIV capture,surfaces were modified with NeutrAvidin. At the end of NeutrAvidinmodification, a statistically significant wavelength peak shift wasobserved (4.4±1.1 nm) (n=6, p<0.05).

To prevent non-specific binding, 10% BSA solution was used as a blockingagent. This blocking agent did not result in a statistically significantpeak shift (n=6, p>0.05).

Then, biotinylated anti-gp120 polyclonal antibody (Ab) was incubated onthe NeutrAvidin-coated surface. The biotinylated anti-gp120 antibodyresulted in a statistically significant wavelength peak shift (6.8±0.5nm) (n=216, p<0.05).

With the assembly of these further layers, it was found that MUA,NeutrAvidin, BSA and Ab collectively shifted the maximum wavelength to551.9±0.5 nm (n=6-216, p<0.05).

Looking at FIGS. 3B and 3C, to evaluate the repeatability of the surfacechemistry, we defined an equation as follows:

${Repeatability} = {\frac{{Mean}\mspace{14mu} {of}\mspace{14mu} {WS}}{{{Mean}\mspace{14mu} {of}\mspace{14mu} {WS}} + {SEM}} \times 100}$

where WS is wavelength shift, and SEM is the standard error of the mean.In the literature, repeatability is defined as closeness of theagreement between the results of the measurements in the same experimentcarried out under the same conditions. Here, repeatability parameter wasdescribed as the percent variation in wavelength shift measurements forthe surface modifications. The parameter was evaluated for bothwavelength and extinction intensity measurements of each surfacemodification. Overall, the repeatability parameter was observed to be55-98% and 85-99% for wavelength and extinction intensity measurements,respectively. These results indicated that the surface chemistrypresented a reproducible, reliable, accurate, repeatable and feasiblenanoplasmonic platform.

To evaluate potential drift and background signal noise due to anypotential unexpected chemical/physical changes or nonspecific binding onthe surface, we assessed the system using whole blood, HIV-spiked wholeblood, and HIV-infected patient samples. The experiments were performedusing HIV subtypes A, B, C, D, E, G, panel, and discarded anonymousHIV-infected patient samples.

The difference between control (i.e., whole blood without HIV),HIV-spiked whole blood samples and HIV-infected patient samples wasevaluated and is presented in FIGS. 3A and 3D. For control measurements,biosensing surfaces functionalized with NeutrAvidin and polyclonalanti-gp120 antibody were used. The addition of unprocessed whole bloodwithout viruses did not result in a statistically significant peak shift(1.2±1.1 nm, n=6, p>0.05).

As evidenced by the shifts in FIG. 3D, the anti-gp120 polyclonalantibody was observed to reliably and repeatably capture multiple virussubtypes (A, B, C, D, E, G, and panel) using the same platform. Wholeblood was spiked with HIV at (6.5±0.6)×10⁵ copies/mL, (8.3±1.3)×10⁵copies/mL, (1.3±0.2)×10⁶ copies/mL, (3.8±1.2)×10⁶ copies/mL,(1.3±0.2)×10⁶ copies/mL, (1.1±0.3)×10⁶ copies/mL and (2.9±0.5)×10⁶copies/mL for subtypes A, B, C, D, E, G, and panel (a mixture ofdifferent HIV subtypes), respectively. Statistically significantwavelength shifts were observed of 9.3±1.2 nm, 5.4±1.1 nm, 4.4±0.5 nm,7.8±1.1 nm, 6.3±0.8 nm, 5.8±0.7 nm and 6.9±1.2 nm for subtypes A, B, C,D, E, G, and panel, respectively (n=6, p<0.05).

To further analyze the experimental data, we constitute curve fittinganalyzing method and compared the original experimental data with thecure fitting results (FIG. 4A-G). After the application of HIV to thenanoplasmonic platform, the representative wavelength shift waspresented in FIG. 4H.

Statistical assessment on the results was performed using non-parametricKruskal-Wallis one-way analysis of variance followed by Mann-Whitney Utest with Bonferroni correction for multiple comparisons. Statisticalsignificance threshold was set at 0.05, p<0.05. Individual p-values forstatistical analyses are presented in Tables 1 and 2 below.

TABLE 1 Set compared p-value AuNP-PLL 0.01 PLL-MUA 0.08 MUA-NA 0.01NA-BSA 0.19 BSA-Ab 0.01 Ab-Control 0.33

TABLE 2 Set compared p-value Control - Subtype A 0.001 Control - SubtypeB 0.001 Control - Subtype C 0.001 Control - Subtype D 0.001 Control -Subtype E 0.001 Control - Subtype G 0.001 Control - Subtype Panel 0.001In FIG. 3A, brackets connecting individual groups indicate statisticallysignificant peak shifts. In FIGS. 3A and 3D, error bars representstandard error of the mean.

Example IV

FIGS. 5A through 5C provide surface characterization of polystyrene (PS)substrates before and after surface modification steps using AtomicForce Microscopy (AFM). Five random 5 μm×5 μm surface areas for eachsample group were evaluated. FIG. 5A shows a PS substrate withoutsurface modification (that is, before the first coating with PLL). FIG.5B shows a PS substrate after 0.05 mg/mL of PLL treatment. FIG. 5C showsthe surface after gold nanoparticles (AuNPs) was immobilized onpoly-l-lysine (PLL) treated surfaces. After AuNP immobilization,nanoparticles were observed on surfaces.

Turning now to FIG. 5D, the root mean square (RMS) roughness of thesurfaces was evaluated and significant difference was observed when goldnanoparticles were immobilized onto the surfaces compared to PSsubstrates with and without PLL (n=8, p<0.05). The surface roughnesschange was evaluated by Asylum-1 MFP-3D AFM System (Asylum ResearchInc., Santa Barbara, Calif.) under tapping mode using a 9±2 nm AFM tipwith a scan rate of 0.5 Hz. Five random 5 μm×5 μm areas on thebiosensing surfaces were evaluated. On the PS substrate, a roughness of2.54±0.17 nm expressed as RMS measurements±SEM was measured. Aftermodification of the surface with 0.05 mg/mL of PLL, the RMS roughnessvalue was observed to be 2.43±0.20 nm. Gold nanoparticle immobilizationon PLL modified surfaces gave a RMS roughness value of 4.65±0.32 nm.

The results presented in FIG. 5D indicates that there was nostatistically significant difference between unmodified and PLL-modifiedpolystyrene surfaces (n=8, p>0.05). However, there was a significantdifference between gold nanoparticle immobilization and unmodifiedpolystyrene surfaces (n=8, p<0.05). Hence, gold nanoparticle immobilizedsurfaces led to significantly greater changes in RMS roughness valuescompared to PLL modified surfaces (n=8, p<0.05). The statisticalanalysis results and p-values obtained using non-parametricKruskal-Wallis one-way analysis of variance followed by Mann-Whitney Utest with Bonferroni correction were also presented in Table 3, below.

TABLE 3 Set compared p-value PS-PLL 0.83 PS-AuNP 0.003 PLL-AuNP 0.003Brackets connecting individual groups in FIG. 5D indicate statisticallysignificant peak shift. Error bars represent standard error of the mean.

To quantitatively evaluate the coating uniformity and density of goldnanoparticles, AFM analysis was performed on the different areas of themodified surfaces. To assess the distribution uniformity of the goldnanoparticles, the following methods were reported using the publicdomain NIH ImageJ software. The images (5 μm×5 μm) were first convertedinto 8-bit images using the public domain NIH ImageJ software, and then,the lower and upper thresholds were adjusted to 50 a.u. and 200 a.u.,respectively. The uniformity is calculated by utilizing this thresholdrange, and the immobilization of gold nanoparticles presented ˜83%uniform coating on the biosensing surface (n=8). Thus, highly uniformand densely coated nanoplasmonic platform was produced. The modifiedsurfaces were incubated at 4° C., and therefore, the evaporation wasprevented. Thus, any coffee ring effects during gold nanoparticleincubation and surface chemistry were not observed.

Gold nanoparticle immobilization and binding on PLL modified surface wasalso evaluated and it was measured that gold nanoparticles boundsequentially onto the modified surface form active areas of 18.97±1.34nm in height.

Example V

After antibody immobilization on the biosensing platform, polystyrenesurfaces that captured HIV from whole blood was cut by a glass cutter,and then, prepared for Scanning Electron Microscopy (SEM) imaging.

Evidence of the reliable capture of HIV particles was shown using aScanning Electron Microscopy (SEM) in FIG. 5E. On the left side of FIG.5E, a scanning electron microscope image of the captured intact viruseswere presented on the antibody immobilized biosensing surface ispresented denoted by arrows. On the right side of FIG. 5E, highermagnification of a captured HIV was imaged and virus diameter wasmeasured as 177.1 nm. The SEM images were taken at 4.7 mm workingdistance and 4.00 kV accelerating voltage.

The SEM images of captured viruses at multiple locations on chip did notshow any aggregation of captured viruses. During testing of whole bloodsamples, the aggregation of viral particles was not observed and this isfurther supported by repeatability of the capture efficiencies withclinical discarded anonymous HIV-infected patient samples.

Coating uniformity and density of gold nanoparticles on PLL modifiedsurfaces were also qualitatively evaluated using SEM. The resultsobtained by SEM qualitatively supported the AFM results, and SEMexperiments demonstrated that gold nanoparticles uniformly distributedon the surface.

Example VI

Biological environment of viruses provide several potential challengingfactors including excessive level of albumin, casein, immunoglobin, andother proteins, which might cause blocking of the antigen-antibodyinteraction and nonspecific binding. This could increase nonspecificbackground signals and interfere with the system performance. Hence,directly detecting viruses from biological samples is a crucial test toevaluate the biosensing performance and robustness of the system.

To evaluate the limit-of-detection and broad applicability of thenanoplasmonic detection platform in biologically relevant systems,various concentrations of multiple subtypes were analyzed spiked inwhole blood and phosphate buffered saline (PBS). The results of PBS arepresented separately below in a different example. In this Example, theresults of multiple HIV subtypes, i.e., A, B, C, D, E, G, and panel,spiked in whole blood are presented. For each sample, the number of HIVcopies/mL for each sample was quantified using by reversetranscription-quantitative polymerase chain reaction (RT-qPCR) andsampling and this viral load was compared to the observed change inwavelength and/or extinction intensity when the sample was placed on thenanoplasmonic detection platform. Establishing a clear correlationbetween the viral load detected by the RT-qPCR, which is known toprovide accurate viral counts, and the observed change in wavelengthand/or extinction intensity detected when a platform carrying the sampleis measured would strongly suggest that measuring the change inwavelength and/or extinction intensity of the sample could be used as aproxy for measurement of the viral load on the sample, assumingsufficient sensitivity.

HIV-1 subtypes A, B, C, D, E, G, and a panel subtype, which consists of6 major globally prevalent strains of genetically and biologicallycharacterized HIV-1 isolates (A, B, C, D, and circulating recombinantforms (CRF01_AE and CRF02_AG)), were obtained from National Institutesof Health (NIH) under AIDS Research and Reference Reagent Program. Thecatalog number of panel is 11259 at NIH AIDS Research and ReferenceReagent Program. Patient samples were obtained from MassachusettsGeneral Hospital, Boston, Mass. All spiked and patient samples weretested with the detection platform. These subtypes were collected fromclinical samples in the United States and Uganda, and cultured inperipheral blood mononuclear cells (PBMCs) using a standard co-cultureprotocol. Briefly, HIV-1 negative PBMCs were first extracted usingFicoll Hypaque density gradient centrifugation (Histopaque 1077 SigmaH8889). After 3-day phytohemagglutinin (PHA) stimulation (0.25 μg/mL),HIV-1 negative PBMCs were co-cultured with HIV-1 infected PBMCs inR20/IL-2 (100 U/mL), which consists of RPMI-1640 (Cellgro® Mediatech10-040-CV) implemented with L-Glutamine (300 mg/mL), 20% heatinactivated fetal bovine serum (FBS; Gemini), penicillin (50 U/mL),streptomycin (50 μg/mL), HEPES buffer (10 mM), and recombinant humaninterleukin-2 (100 U/mL, Roche). The culture was incubated underhumidified 5% CO₂ atmosphere at 37° C., and culture supernatants werereplaced bi-weekly with fresh medium. 3-day PHA stimulated HIV-1infected PBMCs were added once a week, and supernatants were collectedfor p24 testing (Perkin Elmer®, NEK050b). Culture termination wasdetermined by p24 levels (at least 20 ng/mL) in cell-free supernatant,and virus supernatants were stored at −80° C. for the quantification andsampling assays.

In order to quantify the HIV subtypes by RT-qPCR and sampling, multipleHIV subtypes (A, B, C, D, E, G, and panel) were performed in biosafetylevel (BSL)2+ laboratories, and the biosensing platform was evaluated inBSL2+ laboratories after HIV sample was fixed with aparaformaldehyde-containing solution.

To spike HIV in whole blood, HIV subtypes were first quantified byRT-qPCR (Roche COBAS®, Branchburg, N.J.). The samples with a cyclethreshold (CT) value greater than that of the lowest standard point (50copies/mL), which had a CT value of 30-40 (50 PCR cycles were run intotal). The low copies of HIV viral load were earlier reported usingreverse transcription-quantitative polymerase chain reaction (RT-qPCR)systems. Any sample with a positive signal, but with a cycle threshold(CT) value higher than that of the lowest standard point, was reportedwith a viral load value of less than 50 copies/mL.

We measured HIV concentrations of 6.55×10⁸, 6.37×10⁸, 2.09×10⁹, 7×10⁸,8.39×10⁸, 6.53×10⁸ and 1.48×10⁹ copies/mL for subtypes A, B, C, D, E, Gand panel (a mixture of different HIV subtypes), respectively. Panelconsists of HIV subtype A, B, C, D, and circulating recombinant forms(CRF01_AE and CRF02_AG). For HIV stock sample quantification, viruseswere lysed using guanidine isothiocyanate provided in the QIAamp ViralRNA Mini Kit (Qiagen, Valencia, Calif.), and then HIV RNA was extractedaccording to manufacturer's instructions.

To quantify HIV RNA, RT-qPCR was performed. For reverse transcription ofHIV RNA samples, 10 μL of 2× core RT buffer, 2 μL of 10 μM of reverseprimer (5′-GTCTGAGGGATCTCTCTAGTTACCAG-3′), 0.5 μL of AffinityScript(Applied Biosystems, Carlsbad, Calif.), and 7.5 μL of HIV RNA were usedand performed on GeneAmp PCR System 9700 (Applied Biosystems, Carlsbad,Calif.). The reaction was set up to 25° C. for 5 minute, 45° C. for 60minute and 95° C. for 3 minute incubation. For quantification of RNA, 50μL of the master mixture was applied. Master mixture includes 1× corePCR buffer, 0.4 μM of forward primer LTR-F (5′-TAAAGCTTGCCTTGAGTGCT-3′),reverse primer LTR-R2, 0.2 μM of TaqMan probe LTR-P(5′-AGTAGTGTGTGCCCGTCTGTTGTGTG-3′), 2.5 units of SureStart Taqpolymerase, and 10 μL of cDNA template. For the amplification step, 7300Real-Time PCR System (Applied Biosystems, Carlsbad, Calif.) was firstset up to 25° C. for 5 minutes and 95° C. for 10 minutes, and then, 50cycles of 60° C. for a minute, and 95° C. for 30 seconds were performed.The viral load values were obtained by RT-qPCR and repeated at least 3times each sample for each concentration.

Following the viral load quantification and comparison, HIV stocksolutions of A, B, C, D, E, G, and panel subtypes were spiked intounprocessed whole blood for the final concentration varying from 50copies/mL to 1×10⁶ copies/mL. 100 μL of HIV spiked whole blood wasloaded into each antibody immobilized surface of the nanoplasmonicplatform and incubated at 4° C. for an hour. Whole blood samples withoutHIV spiking were used as controls. A second set of RT-qPCR was performedto minimize dilution inaccuracies. The same quantification protocol wasutilized for discarded HIV patient samples.

Before HIV detection using LSPR, bound viruses were immobilized by afixation solution containing paraformaldehyde and incubated at 4° C. foran hour. Following each surface modification step, surfaces were rinsedwith 1×PBS three times to remove blood cells. Since the lowest detectionlimit of RT-qPCR was set to 50 copies/mL, the average of the multiplePCR results that is below 50 copies/mL were reported as ˜50 copies/mL;(e.g., 22±20 copies/mL in HIV subtype A, 44±11 copies/mL in HIV subtypeC and 57±17 copies/mL in HIV subtype E) and plotted with the originaldata point in the wavelength and extinction intensity shift figures forcurve fitting and/or experimental data maximum methods.

As shown in FIG. 6A, in the presence of HIV subtype A, the highest peakshift was observed at 9.3±1.2 nm at (6.5±0.6)×10⁵ copies/mL. Thedetected peak shift decreased with decreasing viral concentration. Forinstance, down to 50 copies/mL HIV viral load resulted in a change of2.9 nm±0.9 nm.

As shown in FIG. 6B, in the presence of HIV subtype B, the highest((8.3±1.3)×10⁵ copies/mL) and lowest (89±6 copies/mL) concentrations ofHIV viral load resulted in 5.4±1.1 nm and 1.9±1.2 nm peak shifts,respectively.

In FIG. 6C, HIV subtype C spiked in blood samples displayed a peak shiftof 4.4±0.5 nm at (1.3±0.2)×10⁶ copies/mL concentration, and 2.1±0.8 nmshift at −50 copies/mL concentration.

As illustrated in FIG. 6D, in the presence of HIV subtype D, thewavelength shifted by 7.8±1.1 nm at (3.8±1.2)×10⁶ copies/mLconcentration, and by 3.3±0.9 nm at 98±39 copies/mL concentration.

FIG. 6E shows that HIV subtype E spiked in blood samples displayed apeak shift of 6.3±0.8 nm at (1.3±0.2)×10⁶ copies/mL concentration, and1.9±0.5 nm shift at ˜50 copies/mL concentration.

FIG. 6F provides the collected data for HIV subtype G. In the presenceof HIV subtype G, the highest peak shift was observed to be 5.8±0.7 nmat (1.1±0.3)×10⁶ copies/mL. The peak shift for 404±54 copies/mL HIVviral load was 3.3 nm±0.6 nm.

FIG. 6G shows that in the presence of HIV subtype panel, the highestpeak shift was observed to be 6.9±1.2 nm at (2.9±0.5)×10⁶ copies/mL. Thepeak shift for 245±101 copies/mL HIV viral load was 1.3±0.7 nm.

The HIV viral load was validated 8 HIV-infected anonymous discardedpatient whole blood samples using the nanoplasmonic platform. Thediscarded HIV patient samples in whole blood were evaluated and it wasobserved that there was 4.3±1.0 nm at 3910±400 copies/mL. The peak shiftdecreased to 2.3±0.7 nm when the lowest concentration (481±73 copies/mL)sample was evaluated. These curve fitting results correlate with theexperimental data maximum method shown in FIG. 7. Thus, in this work weused the curve fitting method in the analysis, using the experimentaldata maximum method to confirm this method.

Here, we analyzed a total of 216 data points for the HIV spiked wholeblood and other 216 data points for their corresponding antibodyreferences. Each data point was subtracted from its own reference. 9wells out of the 216 reference measurements were more than 1 SEM awayfrom the mean. Also, the measurements with these wells gave negativeresults indicating that these wells had issues with the basic surfacechemistry and thus they were eliminated from the analysis. Another 4wells gave negative results below −1 nm when the HIV-spiked whole bloodcurves were subtracted from the antibody reference of the same well.These wells were not included in the analysis, since these results areinconclusive and potentially due to the variations in the surfacechemistry. Additionally, one well gave a very large shift among the 50copies/mL and 100 copies/mL wells (larger than the same subtype'shighest concentration's shift plus its SEM) so it is not included. The10 negative shifts observed ranging between 0 and −0.5 nm, which arebelow the instrumental step size (1 nm) are equivalent to a no-shift.These may be due to the fact that no viruses were captured in the activearea hence these data points were included in the analysis to reflectthe statistical nature of the capture event.

Additionally, the nanoplasmonic extinction spectra of a nanoparticle canbe affected by neighboring nanoparticles. This property has been used inmeasuring the length of flexible single-strand DNA strains positioned inbetween nanoparticle pairs. It has been shown that short inter-particledistances can amplify the local electromagnetic fields causing anincreased LSPR signal. These enhanced fields (hot-spots) have beenrecently used in nanoplasmonic detection of Adenovirus particles. Theexact nature of this enhancement is reported to be a function of thenanoparticle arrangement on the surface and the effective refractiveindex distribution over nanoparticles.

Example VII

As a second analyzing method, the experimental data maximum was used. Inthe presence of HIV subtype A, the highest peak shift was observed at5.2±0.5 nm at (6.5±0.6)×10⁵ copies/mL (FIG. 7A). The detected peak shiftdecreased with decreasing viral concentration. For instance, down to 50copies/mL HIV viral load resulted in a change of 1.3±0.5 nm (FIG. 7A).In the presence of HIV subtype B, the highest ((8.3±1.3)×10⁵ copies/mL)and lowest (89±6 copies/mL) concentrations of HIV viral load resulted in4.7±0.5 nm and 2.5±0.8 nm peak shifts, respectively (FIG. 7B). HIVsubtype C spiked in blood samples displayed a peak shift of 6.3±0.9 nmat (1.3±0.2)×10⁶ copies/mL concentration, and 2.7±0.9 nm shift at −50copies/mL concentration (FIG. 7C). In the presence of HIV subtype D, thewavelength shifted by 4.3±0.5 nm at (3.8±1.2)×10⁶ copies/mLconcentration, and by 1.7±0.5 nm at 98±39 copies/mL concentration (FIG.7D). HIV subtype E spiked in blood samples displayed a peak shift of4.7±0.8 nm at (1.3±0.2)×10⁶ copies/mL concentration, and 1.2±0.8 nmshift at −50 copies/mL concentration (FIG. 7E). In the presence of HIVsubtype G, the highest peak shift was observed to be 4.8±0.9 nm at(1.1±0.3)×10⁶ copies/mL (FIG. 7F). The peak shift for 404±54 copies/mLHIV viral load was 2.0±0.7 nm (FIG. 7F). In the presence of HIV subtypepanel, the highest peak shift was observed to be 6.5±0.8 nm at(2.9±0.5)×10⁶ copies/mL (FIG. 7G). The peak shift for 245±101 copies/mLHIV viral load was 1.5±0.5 nm (FIG. 7G). We also validated HIV viralload using 9 HIV-infected anonymous discarded patient whole bloodsamples using the nanoplasmonic platform. In the presence of discardedpatient samples, the highest peak shift was observed to be 3.0±0.6 nm at4169±578 copies/mL (FIG. 7H). The peak shift for 481±73 copies/mL HIVviral load was 1.3±0.6 nm (FIG. 7H).

In addition to the wavelength shift, the nanoplasmonic platform can beused to measure extinction intensity variations caused by HIV captureand detection events. The capture events cause both wavelength shift andextinction intensity variation, and the results were acquired both aswavelength shifts and extinction intensity variations. The extinctionintensity variations producing the reported detection capability wereevaluated for the same set of experiments as in the wavelength shiftplots of FIGS. 7A through 7G. In FIGS. 8A though 8H, the extinctionintensity variations are compared to the viral load detected by RT-qPCR.

In FIG. 8A, HIV subtype A in whole blood led to a 0.068±0.004 a.u.extinction intensity shift at (6.5±0.6)×10⁵ copies/mL. The extinctionintensity shift was observed to be 0.029±0.006 a.u. when the lowestconcentration (down to 50 copies/mL) sample was used.

In FIG. 8B, (8.3±1.3)×10⁵ copies/mL of HIV subtype B spiked in wholeblood samples led to an extinction intensity shift of 0.070±0.003 a.u.The extinction intensity shift decreased to 0.030±0.004 a.u., when 89±6copies/mL HIV concentration was evaluated.

In FIG. 8C, for sampling with HIV subtype C in whole blood,(1.3±0.2)×10⁶ copies/mL concentration was evaluated and the extinctionintensity shift was observed to be 0.071±0.005 a.u. At ˜50 copies/mLconcentration, 0.031±0.003 a.u. extinction intensity shift was observed.

In FIG. 8D, (3.8±1.2)×10⁶ copies/mL of HIV subtype D in whole bloodsample resulted in an extinction intensity shift of 0.059±0.003 a.u. Ata low concentration (98±39 copies/mL), 0.024±0.003 a.u. extinctionintensity shift was observed.

In FIG. 8E, HIV subtype E in whole blood was evaluated and it wasobserved that there was 0.057±0.003 a.u. extinction intensity shift at(1.3±0.2)×10⁶ copies/mL. The extinction intensity shift decreased to0.029±0.007 a.u. when the lowest concentration (˜50 copies/mL) samplewas used.

In FIG. 8F, for sampling with HIV subtype G in whole blood,(1.1±0.3)×10⁶ copies/mL concentration was evaluated and the extinctionintensity shift was observed to be 0.059±0.003 a.u. At 404±54 copies/mLconcentration, 0.030±0.003 a.u. extinction intensity shift was observed.

In FIG. 8G, HIV subtype panel in whole blood was evaluated and it wasobserved that there was 0.059±0.002 a.u. extinction intensity change at((2.9±0.5)×10⁶ copies/mL. The extinction intensity shift decreased to0.032±0.002 a.u. when the lowest concentration (245±101 copies/mL)sample was used.

In FIG. 8H, discarded HIV patient samples in whole blood were evaluatedand it was observed that there was 0.034±0.003 a.u. extinction intensityshift at 4169±578 copies/mL. The extinction intensity shift decreased to0.023±0.002 a.u. when the lowest concentration (481±73 copies/mL) samplewas evaluated.

Example VIII

To evaluate the repeatability of the biosensing platform technology, anequation for a repeatability parameter was defined as follows:

${Repeatability} = {\frac{{Sum}\mspace{14mu} {of}\mspace{14mu} {WS}\mspace{14mu} {per}\mspace{14mu} {concentration}}{{{Sum}\mspace{14mu} {of}\mspace{14mu} {WS}\mspace{14mu} {per}\mspace{14mu} {concentration}} + {{SEM}\mspace{14mu} {per}\mspace{14mu} {concentration}}} \times 100}$

where WS is wavelength shift, and SEM is the standard error of the mean.In the literature, repeatability is defined as closeness of theagreement between the results of the measurements in the same experimentcarried out under the same conditions. Here, repeatability parameter wasdescribed as the percent variation in wavelength shift measurements forthe same virus concentration.

The parameter was evaluated for each HIV subtype, and presented atvarying spiked sample concentrations. Overall, the repeatabilityparameter was observed to be 56-90% for a broad range of concentrationsfor multiple HIV subtypes and discarded HIV patient samples. Theseresults indicated that the nanoplasmonic biosensing platform isreliable, accurate, repeatable and feasible. These results alsodemonstrated that the system performance was independent of the subtypeand concentration, and showed comparable repeatability values formultiple HIV subtypes in whole blood.

The specific repeatability results are as follows for curve fittinganalysis. In HIV subtype A spiked samples, the repeatability wasobserved to be 76-89% at the corresponding concentrations ranging from˜50 copies/mL to (6.5±0.6)×10⁵ copies/mL. In HIV subtype B spikedsamples, the repeatability was observed to be 64-83% at thecorresponding concentrations ranging from 89±6 copies/mL to(8.3±1.3)×10⁵ copies/mL. In HIV subtype C spiked samples, therepeatability was observed to be 71-89% at the correspondingconcentrations ranging from ˜50 copies/mL to (1.3±0.2)×10⁶ copies/mL. InHIV subtype D spiked samples, the repeatability was observed to be78-90% at the corresponding concentrations ranging from 98±39 copies/mLto (3.8±1.2)×10⁶ copies/mL. In HIV subtype E spiked samples, therepeatability was observed to be 72-90% at the correspondingconcentrations ranging from ˜50 copies/mL to (1.3±0.2)×10⁶ copies/mL. InHIV subtype G spiked samples, the repeatability was observed to be74-89% at the corresponding concentrations ranging from 404±54 copies/mLto (1.1±0.3)×10⁶ copies/mL. In HIV subtype panel spiked samples, therepeatability was observed to be 56-85% at the correspondingconcentrations ranging from 245±101 copies/mL to (2.9±0.5)×10⁶copies/mL. In HIV patient samples, the repeatability was observed to be59-81% at the corresponding concentrations ranging from 481±73 copies/mLto 4169±578 copies/mL.

With reference to FIG. 8I, the repeatability parameter was evaluated forthe extinction intensity shifts for multiple HIV subtypes at variousconcentrations. To do this, the equation above would be modified toreplace the sum of wavelength shift per concentration with the sum ofthe extinction intensity shift per concentration. Overall, therepeatability parameter was observed to be 83-99% for a broad range ofconcentrations for multiple HIV spiked blood and discarded HIV-infectedpatient samples. The system performance was independent of the subtype(n=4-6, error bars represent standard error of the mean). The viral loadvalues were obtained by RT-qPCR and repeated at least 3 times eachsample for each concentration.

Example IX

An HIV subtype panel suspension in phosphate buffered saline (PBS) wasalso evaluated using SPR and RT-qPCR and the results are reported inFIG. 9. There was 5.9±1.1 nm wavelength shift at ((7±0.4)×10⁶ copies/mL.The peak shift decreased to 1.0±0.5 nm when the lowest concentration(138±10 copies/mL) sample was used (Error bars represent standard errorof the mean). We performed 3 replicates for RT-qPCR measurements and 6replicates for nanoplasmonic measurements for each samples.

Example X

To evaluate quantitative detection, the standard curve with HIV-spikedwhole blood samples was obtained, using the wavelength shifts and theHIV viral load obtained by RT-qPCR. After the standard curve wasobtained, quantitative detection results using the biosensing platformwith HIV-infected patient samples were obtained and are presented inFIGS. 10A and 10B and Table 4.

Turning first to FIG. 10A, quantitative detection results are providedfor HIV-infected patient samples. In FIG. 10A, discarded HIV infectedpatient samples in whole blood were evaluated using the nanoplasmonicplatform and correlation was presented between HIV count obtained byRT-qPCR and the nanoplasmonic platform. The nanoplasmonic platformpresented a viral load from (1.3±0.7) log₁₀ (copies/mL) to (4.3±1.2)log₁₀ (copies/mL) in patient samples. RT-qPCR count presented a viralload from (2.7±0.1) log₁₀ (copies/mL) to (3.6±0.1) log₁₀ (copies/mL) inpatient samples. Quantitative measurements of HIV infected patientsamples using both methods are presented in Table 4 below.

TABLE 4 RT-qPCR Count Nanoplasmonic Count (Mean ± SEM) (Mean ± SEM)Patient # (log₁₀ copies/mL) (log₁₀ copies/mL) I 3.6 ± 0.1 4.3 ± 1.2 II3.3 ± 0.1 3.3 ± 0.9 III 2.9 ± 0.2 2.2 ± 1.1 IV 2.8 ± 0.1 2.6 ± 1.2 V 2.7± 0.1 2.0 ± 0.8 VI 3.0 ± 0.1 2.0 ± 0.8 VII 2.8 ± 0.1 2.5 ± 1.0 VIII 2.9± 0.1 1.3 ± 0.7

In FIG. 10B, Bland-Altman Analysis was performed between thenanoplasmonic platform and RT-qPCR counts and did not display anevidence for a systematic bias for HIV viral load for HIV-infectedpatient blood samples tested.

Example XI

Having established above that HIV viral loads can be captured on abiosensing surface of a nanoplasmonic platform, and then, detected usinglocalized surface plasmon resonance, the next steps for achievingportability for POC detection include implementing this nanoplasmonicplatform or an analog structure on a microchip, and then, confirmingthat this microchip provides similar results. The disposable andportable microchip device makes it possible to perform testing at remotesettings where access to instrumentation for such analyses is eitherunavailable or expensive.

With additional reference to process steps depicted in FIG. 11 and themicrochip structure depicted in FIG. 12C, the steps for viral loaddetection on a microchip are generally described. In steps 1 and 2, awhole blood sample is obtained from a patient and is placed on themicrochip. Approximately a 100 μL of fingerprick volume of blood shouldbe sufficient. This blood sample is passed through a microfluidiccapture channel in which red blood cells and white blood cells areremoved on-chip by filtering. Then in the capture channel depicted inFIG. 12C, HIV-1 in whole blood will be captured using immobilizedcapture agents on microchannel surface immobilized with metalnanoparticles that are coated by anti-gp120 antibody as is generallydepicted in steps 2 and 3 of FIG. 11. In step 4, the captured HIV on themicrochip is detected and quantified using a portable CCD-basedlocalized surface plasmon resonance technology.

Example XII

With particular reference to FIG. 12, a microfluidic device of the typedescribed in the previous example can be constructed in the followingway. As best shown in FIG. 12C, the microfluidic device can comprises(i) a passive filtration chamber (e.g., a micromachined polycarbonatefilter) that removes red blood cells and white blood cells and (ii) afollowing chamber that captures HIV from the filtered plasma. Thecapture chamber of the microchip can be constructed with polystyrene,glass parylene, quartz crystal, graphene and mica layers,polydimethylsiloxane (PDMS), paper, and poly(methyl methacrylate) (PMMA)and double-sided adhesive film as depicted in FIG. 12A.

In the depicted embodiment, a simple channel design is employed with afilter inserted in the middle, which has been characterized with respectto whole blood properties in our other studies. However, in otheralternative embodiments, the device or interfaced devices may havetubing that directly connecting chambers or by building the filters intochannels.

Virus capture efficiency can be increased by removing the cells fromblood using a filter approach. In the chip design, the filter (0.2-3 μmpore size) is placed at the inlet and plasma passing through the filteris directly introduced into the capture chamber. Preferably, red bloodcells and white blood cell will not be passing through the filter below2 μm pore size. This approach will help to capture HIV-1 with higherefficiency in the microchip.

The device fabrication is technically simple involving layered stackingof PMMA, double-sided adhesive film and a glass cover as depicted inFIGS. 12A and 12B, which can be easily scaled up for batch production.The PMMA base has a dimension of 24×40×3.175 mm³ and it is cut using alaser cutter (VersaLaser, VLS2.3). On the PMMA base, an inlet and anoutlet are cut out for injecting samples and collecting waste,respectively. The channel is cut into double sided adhesive (DSA) filmby the laser cutter. Prior to assembling, surface of the glass coverslipis modified by plasma treatment. The coverslip is then immediatelymounted onto the DSA film, which is previously assembled with the PMMAbase. After assembling these three components, a microchannel is formed.FIG. 12C shows a schematic of a viral load microchip, which can befabricated using machining and molding processes. The microchip is madewith multiple pieces to ease the integration, assembly, and packaging.

The Hele-Shaw flow theory and design equations can be used to determinethe flow rates and shear stress on the surface based on microchannelgeometry and dimensions. By targeting optimum shear rates and flow ratesfor capture, the microfluidic channel dimensions on the microchip can bedesigned accordingly. The longer and wider the microchip, the moresurface is available to capture HIV. The channel height can be made assmall as 10 μm, which then would allow viruses to interact more with thefunctionalized surface. For various channel dimensions, typical flowrates will be in the range of 5-20 μL/min. Preferably, for a rapid testthe flow rate should be adjusted to be as high as possible (>5 μL/min)without significantly compromising the capture efficiency.

A further modification to the design illustrated in FIG. 12A can includereplacing the glass with non-sharp materials to enhance handling safety.For example, the glass slide may be replaced by parylene or PMMA asthese materials are optically transparent and can be functionalized withantibodies.

Example XIII

To capture HIV-1 from fingerprick blood, the microchannels can befunctionalized with anti-gp120 antibody as described above with respectto FIG. 1. In addition to anti-gp120 antibody, microchips can be builtwith various other capture agents including anti-gp41 antibody,anti-gp24 antibody, lectin and/or sCD4.

Although various concentrations of PLL and gold nanoparticles may beused, in order to optimize surface chemistry for maximum detectionefficiency, it is believed PLL concentration should generally be in therange of 0.01 mg/ml to 1 mg/ml to immobilized nanoparticles in the rangeof 1.14×10¹¹ to 5.7×10¹¹ particles per mL onto the microchip. It shouldbe appreciated that various nanoparticle concentrations and sizes(10-100 nm) might be used and that optimal PLL concentrations andnanoparticle concentrations may vary based on the nanoparticle size. Asdescribed in the examples above, NeutrAvidin is covalently coupled withNHS ester and EDC by incubating the microchannel surface withNeutrAvidin (10 μg/mL in PBS) for 1 h at 4° C. Finally, capture agentsolution (e.g., biotinylated anti-gp120, 40 μg/mL in PBS containing 1%(w/v) BSA and 0.09% (w/v) sodium azide) is injected to react at 4° C.for 1 hr. After each step, surfaces can be rinsed with PBS to wash awayunreacted molecules. The chips can be stored at 4° C. for up to 3 monthswithout loss of function.

Example XIV

To perform localized surface plasmon resonance, a portable lenslessCCD-based optical system is used with the microchips for rapid virusdetection and automatic quantification by LSPR. Briefly, HIV-1 fromfingerprick blood is captured when flowed in the microchannelfunctionalized with capture agents. This HIV can be repeatedly andreliably captured with high sensitivity across a range of values thatinclude the current WHO definition of treatment failure (VL>5,000copies/mL) as well as the DHHS and ACTG definitions of treatment failure(VL>200 copies/mL).

For LSPR detection, wavelength shift or extinction intensity change onthe entire channel surface of the chip can be detected by the aid of aCCD sensor (KODAK, KAI-11002, Rochester, N.Y.) carrying 11 millionsquare pixels (9 μm wide) as illustrated in step 4 of FIG. 12. Theactive CCD sensor area is 37.25×25.70 mm. A portable spectrometer, whichscans spectra from 400 nm to 800 nm is used to illuminate active area ofthe microchip. Once HIV is captured on the active area of the capturedetection channel, changes at the close vicinity of the active surfacecauses a shift in the wavelength or extinction intensity. This shift isdetected instantly within 10 seconds by intensity change of the recordedCCD image quantifying the captured viruses. The wavelength shift and/orextinction intensity change is then quantified automatically with theaid of in-house signal processing software such as MATLAB within another20 seconds.

To summarize, the system detects intensity variations on the CCDresulting from changes of nanoparticle's specific wavelength in closevicinity of active surface quantifying the HIV-1 viral load from wholeblood.

HIV can be detected repeatably and reliably with high sensitivitycovering the range of values that include the current WHO definition oftreatment failure (VL>5,000 copies/mL) as well as the DHHS and ACTGdefinitions of treatment failure (VL>200 copies/mL).

With further scaling, it is contemplated that the viral load in 100 μLof whole blood can be analyzed in as little as 15 minutes using a systemof this type. By controlling parameters such as flow rates, surfacechemistry and channel size, capture, detection and assay duration can,within certain limits, be controlled. For instance, it takes 10 minutes(at 10 μL/minute flow rate) to run 100 μL of blood into a microchannelto capture HIV, and an additional 4 minutes to wash the channels (at 20μL/minute flow rate). It takes an additional 30 seconds to obtain aviral load using the sensor platform by running automated software. A15-minute viral load is anticipated with a satisfactory throughputperformance at the POC, when compared to existing systems (e.g.reverse-transcriptase activity test: 3 days) and RT-qPCR (one day).

Example XV

An example is now provided which describes the construction of aflexible nanoplasmonic platform.

To construct a flexible platform, polyester surfaces were used as a basematerial for localized surface plasmon resonance (LSPR) experiment. Thesurfaces were first modified with poly-l-lysine (PLL) (although otherlayers might be able to immobilize the nanoparticles on the substrateand not interfere with LSPR detection). PLL provided amine groups toimmobilize gold nanoparticles on the substrate. The nanoplasmonicimmobilization layer was then functionalized with thiol groups, althoughother attachments between the layer and the substrate or the goldnanoparticles might also be employed as long as it did not impair theability of the nanoplasmonic platform to be read using LSPR.

To link the one or more surface recognition elements such as antibodiesand capture moieties to the gold nanoparticles, a modified surface wasformed by preparing a surface of the plurality of gold nanoparticlesusing a mercaptoundecanoic acid to form carboxyl groups, reactingN-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride with thecarboxyl groups to form an amine reactive intermediate, and stabilizingthe amine reactive intermediate by N-hydroxysulfosuccinimide to form themodified support surface. Then, NeutrAvidin protein was used toimmobilize biotinylated anti-gp120 polyclonal antibody. Additionally,other or additional antibodies may be present based on the pathogens andinfectious agents to be detected. It is contemplated that multiplepathogens may be detected on a single flexible nanoplasmonic platform.In some embodiments, the antibody may be a polyclonal antibody. In thecontext of HIV detection, the antibody and/or capture moieties may be,for example, a gp120 antibody, a gp41 antibody, a gp24 antibody, solubleCD4, or lectin, all of which are able to detect multiple subtypes ofHIV. Additionally, in some forms, the modified support surface is linkedto at least one of a protein A, a protein G, a protein A/G, aStreptavidin protein, and a NeutrAvidin protein which is used to formchemical bonds to immobilize recognition elements such as the antibodyon the modified support surface.

As outlined above, after the gold nanoparticle coating onto the PLLmodified surface, MUA was self-assembled as a monolayer onto the goldnanoparticle layer. Antibodies immobilized with a favorable orientationusing NeutrAvidin have a higher capture efficiency towards bioagentsthan those immobilized by physical absorption and chemical binding ofantibodies onto a surface. To immobilize biotinylated antibodies for HIVcapture, surfaces were modified with NeutrAvidin. To preventnon-specific binding, 10% BSA solution was used as a blocking agent.Then, biotinylated anti-gp120 polyclonal antibody (Ab) was incubated onthe NeutrAvidin-coated surface. After each surface modification step,the surfaces were rinsed with 1×PBS three times to remove unboundchemical moieties. With the assembly of these further layers, it wasobserved that MUA, NeutrAvidin, BSA and Ab collectively shifted themaximum wavelength to 543.5±0.3 nm as is illustrated in FIG. 13. Here, aflexible nanoplasmonic surface was constructed, and this platformpresented comparable results with the previously describedpolystyrene-based nanoplasmonic technology.

Example XVI

This example describes the use of disposable chips to detectantiepileptic drug (AED) serum concentrations at the point of care usingthe nanoplasmonic platform.

Epilepsy is a neurological disorder characterized by brief episodes ofinvoluntary shaking sometimes accompanied by loss of consciousness andcontrol of bowel or bladder function. About 88% of patients havereported drug related side-effects including restlessness, feeling ofaggression, and suicide associated with depression. Most of these sideeffects are dose dependant.

AEDs are the mainstay of treatment for persons with epilepsy (PWE).However, as many as two out of three PWE suffer from recurrent seizuresor AED-related side effects. In either case, the optimization of AEDdosages is clinically important and often guided by measuring AED serumconcentrations. These side-effects can be minimized by adjusting theantiepileptic drug (AED) dosage and timing of the dosage. Monitoring ofthe AED serum concentrations can guide the AED optimization goal.Currently, the blood tests used for AED serum concentrations are labbased and cannot be performed at home or at the time of seizures, andthus, obtaining blood tests is presently impractical due to theassociated inconvenience (lab based detection) and costs, as well as theoften long latency between side-effects and/or seizures and when bloodtests are obtained.

AED serum concentrations are conventionally quantified by HighPerformance Liquid Chromatography (HPLC) in conjunction with UV andphotodiode arrays as illustrated by the top path in FIG. 14. However,these methods are lab-based and not point-of-care nor obtained at themoment of need (for example after a seizure or when side effects areoccurring, because they rely on expensive equipment and skilledtechnicians.

Now, a microfluidic-based disposable AED detection system is describedthat can be performed anywhere and automated to handle 10-100 μL ofblood obtained with a fingerprick, such as used for blood glucosemonitoring. Rather than blood, it is contemplated that other bodilyfluids such as urine, sweat and saliva could also be collected andanalyzed. The glass surface coated with gold nanoparticles isfunctionalized with anti-AED antibody for specific capture of AEDmolecules. Upon drug-Ab binding, a shift in the extinction intensity andwavelength spectrum can be monitored due to localized surface plasmonresonance effect of gold nanoparticles. The system consists of aportable spectrophotometer for detection and presents the results inapproximately 10 minutes. A version of this device may be made withread-outs that the patient or family member can monitor, to report tothe physician or to implement actions that the physician provided themin advance. This new microfluidic-based disposable AED detection systemis illustrated on the bottom path of FIG. 14 and satisfies the need fora simple, rapid, reliable and disposable test for AED measurements thatcan be performed at the doctor's office or by the patient or a familymember at home.

In the development of this microfluidic-based disposable AED detectionsystem, two interdependent, distinct specific aims are met. First,enzyme-linked immunosorbent assay (ELISA) is translated to 10 minuteslocalized surface plasmon resonance (LSPR) microchip device for thedetection of AED serum concentrations, starting with AEDs that havenarrow therapeutic windows. A laptop/tablet application may be used tosend results to doctor's office. A microchip device is provided containsmultiple microchannels whereby serum concentrations of various AEDs aresimultaneously detected for patients on polytherapy, as well as othercomponents of whole blood, such as WBC counts. Second, the device isvalidated with sufficient numbers of discarded anonymous clinicalsamples to demonstrate acceptable sensitivity and specificity.Bland-Altman analysis is performed to statistically compare the LSPRresults with HPLC (i.e., gold standard method).

The disposable LSPR microchip employs a gold nanoparticle coated surfacefunctionalized with anti-AED antibody to quantify the drug amount. Aportable spectrophotometer is used for spectrum analysis for theread-out, which can then be sent automatically to the doctor's office ifthe test is done elsewhere. The disposable microchip may be inexpensive(for example, less than $2), may provide results within 10 minutes, maybe stable at room temperature, and may be used by patients andhealth-care providers.

The design of the proposed LSPR microfluidic assay for thequantification of AED in a blood serum is illustrated in FIG. 15. Theanti-Carbamapezine (CBZ) antibody is attached to the gold nanoparticles.Anti-CBZ antibody specifically captures the CBZ present in the bloodserum. When the chip surface is analyzed using a spectrophotometer, thegold nanoparticles produce the extinction intensity spectrum due totheir enhanced nanoplasmonic properties with peak intensity at specificwavelength. This Anti-CBZ antibody capture events quantitatively shiftthe peak wavelength (that is, the higher the concentration of AED inblood serum, the higher is the shift in the peak wavelength).

The nanoplasmonic platform technology described above for the intact HIVdetection from unprocessed whole blood can be adapted for quantificationof AED in a blood serum. This platform allows efficient capture ofintact HIV from whole blood by immobilized gold nanoparticles, resultingin a shift in the peak of the maximum extinction wavelength. Thisdetection technology utilizes from LSPR signals of metal nanoparticlesbased on the changes in collective oscillations of free electronssurrounding the nanoparticles. For producing the nanoplasmonic platform,gold nanoparticles were first immobilized on polystyrene surfaces usingpoly-L-lysine (PLL) as illustrated in FIG. 1. After the activation ofgold surfaces with several chemicals and activators(11-Mercaptoundecanoic acid (MUA), N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide (NHS)),anti-gp120 polyclonal antibodies were immobilized for the capture ofintact HIV from whole blood samples. To analyze the wavelength andextinction intensity data, a curve fitting method was developed, whichwas written using MATLAB software to find the nanoplasmonic wavelengthpeak of each recorded experimental spectra. Then, multiple HIV subtypes(i.e., A, B, C, D, E, G, and panel (a mixture HIV subtype A, B, C, D,and circulating recombinant forms (CRF01_AE and CRF02_AG)) spiked inwhole blood were evaluated as a model detection platform. Variousconcentrations of multiple HIV subtypes, i.e., A, B, C, D, E, G, andpanel, spiked in whole blood ranging from 50 to (3.8±1.2)×10⁶ copies/mLwere assessed as illustrated in FIG. 16A, which shows the aggregateddata from FIGS. 6A-6H. Limit-of-detection (LOD) for each subtype wasdetermined as a statistically significant peak shift compared to thecontrol (i.e., whole blood without HIV) samples. LOD was observed as1,346±257 copies/mL for HIV subtype A, 10,609±2,744 copies/mL for HIVsubtype B, 14,942±1,366 copies/mL for HIV subtype C, 98±39 copies/mL forHIV subtype D, 120,159±15,368 copies/mL for HIV subtype E, 404±54copies/mL for HIV subtype G, and 661±207 copies/mL for HIV subtype panelas illustrated in FIG. 16B. These results demonstrated that thepresented nanoplasmonic platform can detect and capture intact HIV fromunprocessed whole blood samples with a sensitivity of down to 98±39copies/mL. The platform was further validated with 8 HIV-infectedanonymous discarded patient whole blood samples. The patient samplespresented wavelength shifts from 2.3±0.7 nm to 4.3±1.0 nm for (1.3±0.7)log₁₀ copies/mL to (4.3±1.2) log₁₀ copies/mL, respectively asillustrated in FIG. 10A. To evaluate quantitative detection, a standardcurve was obtained from HIV spiked whole blood samples using thewavelength shifts from the nanoplasmonic response and the HIV viral loadobtained by RT-qPCR (gold standard method). Bland-Altman comparisonanalysis was used to evaluate the repeatability of the nanoplasmonicplatform count using residual analysis in comparison to RT-qPCR counts.The results agreed with the RT-qPCR results in the clinically acceptablerange, and there was no evidence observed for a systematic bias for HIVviral load for tested HIV-infected patient blood samples as illustratedin FIG. 10B. These results indicated that the nanoplasmonic biosensingplatform is reliable, accurate, repeatable and feasible for smallstructures such as virus detection without any sample preprocessing.This presented platform technology can be potentially used as a broadlyapplicable tool to diagnose other clinical diseases or to monitortreatment efficacy in resource-constrained settings. Additionally, thisplatform technology can be used to detect oncoviruses as well.

For AED measurement, a similar microfluidic based LSPR setup may be usedto that described above for HIV viral load quantification.

In some embodiments, the microfluidic channel is designed by sandwichingdouble-sided adhesive film (DSA) (50 μm thick; obtained from iTapestoreof Scotch Plains, N.J.) between glass slide and poly(methylmethacrylate) (PMMA) (3 mm thick; obtained from McMaster Carr ofAtlanta, Ga.). Both of the DSA and PMMA were cut using a laser cutter(Versa Laser™, AZ).

In some embodiments, the surface chemistry starts with the surfaceimmobilization of gold nanoparticles inside the microfluidic channelsusing PLL. After the activation of gold surfaces with several chemicalsand activators (MUA, EDC, andNHS), NeutrAvidin is immobilized followedby conjugation with biotinylated anti-CBZ monoclonal antibodies. Serumsamples spiked with CBZ at various concentrations are tested by usingthese chips. The chips are analyzed using portable spectrophotometer andCCD sensor.

It is contemplated that the microfluidic devices may be integrated withdisposable cartridges. FIG. 17 schematically demonstrates a schematic ofsuch cartridge, which can be fabricated using machining and moldingprocesses. The cartridge can be made with multiple pieces to ease theintegration, assembly, and packaging of the microchip. The chip may befunctionalized before being placed into the cartridge. The microfluidicchips can be disposable for ease of use and to avoid contamination. Theymight be made of glass bonded to glass or plastic with an adhesive tomake the chip easy to fabricate. The material cost to build such achannel could be less than $2 for batch, although the material costshould in no way be considered limiting.

Example XVII

In this example, the capture and detection of Escherichia coli (E. coli)using the nanoplasmonic platform technology are described. As mentionedabove, other bacteria as well as fungi or yeast could also be capturedand detected using this platform.

The surface chemistry for E. coli capture and detection was started withthe modification of the polystyrene substrates with poly-l-lysine (PLL)(although other layers might also be able to immobilize thenanoparticles on the substrate and not interfere with LSPR detection).PLL provided amine groups to immobilize gold nanoparticles on thesubstrate. The nanoplasmonic immobilization layer was thenfunctionalized with thiol groups, although other attachments between thelayer and the substrate or the gold nanoparticles might also be employedas long as it did not impair the ability of the nanoplasmonic platformto be read using LSPR.

To link the one or more surface recognition elements such as antibodiesand capture moieties to the gold nanoparticles, a modified supportsurface was formed by preparing a surface of the plurality of goldnanoparticles using a mercaptoundecanoic acid to form carboxyl groups,reacting N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloridewith the carboxyl groups to form an amine reactive intermediate, andstabilizing the amine reactive intermediate by N-hydroxysulfosuccinimideto form the modified support surface. Then, NeutrAvidin protein was usedto immobilize anti-Lipopolysaccharide Binding Protein (LBP) antibody. E.coli surface has a specific polysaccharide called lipopolysaccharide(LPS), and thus, LBP was linked to the anti-LBP antibody for theefficient capture and detection of E. coli. The anti-LBP antibody allowsLBP to gain its favorable orientation for the efficient capture anddetection. Additionally, other or additional antibodies may be presentbased on the pathogens and infectious agents to be detected. It iscontemplated that multiple pathogens may be detected on a singlenanoplasmonic platform. A multiplexed detection platform with parallelchannels is also contemplated. In some embodiments, the antibody may bea polyclonal antibody. In the context of E. coli detection, the antibodyand/or capture moieties may be, for example, an anti-LPS antibody, LBP,cluster of differentiation 14 protein (CD14) of human monocyte andanti-flagellin antibody, all of which are able to detect E. coli.Additionally, in some forms, the modified support surface is linked toat least one of a protein A, a protein G, a protein A/G, a Streptavidinprotein, and a NeutrAvidin protein which is used to form chemical bondsto immobilize recognition elements such as the antibody on the modifiedsupport surface.

In the experiments, E. coli strain BL21 Star™ was used, and culturedonto Luria-Bertani (LB) agar plates containing 100 mg/mL of ampicillin.Then, the sample was incubated at 37° C. for 16 hours. An isolated E.coli colony was picked from the plate and inoculated in 5 mL of LBmedium on another LB agar plate containing 100 mg/mL of ampicillin. TheE. coli culture was transferred and incubated at 37° C. with shaking at250 rpm for 16 hours, and then, aliquoted as a standard stock for allexperiments. After that, the stock solution was diluted ten-fold inphosphate buffered saline (PBS) and spread on LB-ampicillin plates.After overnight incubation at 37° C., single colonies of E. coli werequantified, and the number of the original concentration of the stockwas calculated.

Following the quantification of the stock sample, E. coli spiked in PBSsamples were prepared for the final concentration ranging from 10⁴ to10⁸ CFUs/mL. 100 μL of the E. coli spiked sample was loaded into eachantibody immobilized surface of the nanoplasmonic platform and incubatedat 4° C. for an hour. PBS samples without E. coli spiking were used ascontrols. Following each surface modification step, the surfaces wererinsed with 1×PBS three times to remove unbound bacteria and chemicalmoieties. In the presence of E. coli, the peak of wavelength at previousstep (that is, LBP binding) shifted as shown in FIG. 18. In the selecteddetection range, the highest detected peak shift was observed as 9.0nm±0.4 nm at 10⁸ CFUs/mL concentration as shown in FIG. 19A. The peakshift decreased with decreasing E. coli concentration, and the lowestshift was observed as 2.5 nm±0.6 nm at 10⁴ CFUs/mL concentration as alsoshown in FIG. 19A. Additionally, the repeatability parameter wasobserved to be 82-96% for E. coli capture and detection experimentsusing nanoplasmonic platform as illustrated in FIG. 19B. Here, areliable, repeatable, feasible, label-free, and fluorescence-free andrepeatable technology is presented that captures and detects E. colifrom biologically relevant solutions such as PBS, blood, urine, andsaliva.

Example XVIII

In this example, the capture and detection of interferon-γ usingnanoplasmonic platform technology is described.

The quantification of the released interferon-γ (IFN-γ) has been used toevaluate the status of tuberculosis (TB) patients. T cells release IFN-γas a host cellular immune response upon the stimulation of whole bloodwith TB-specific antigens.

To construct a nanoplasmonic platform for the IFN-γ capture anddetection, polystyrene surfaces were first modified with poly-l-lysine(PLL). Here, PLL provided amine groups to immobilize gold nanoparticleson the substrate. The nanoplasmonic immobilization layer was thenfunctionalized with thiol groups, although other attachments between thelayer and the substrate or the gold nanoparticles might also be employedas long as it did not impair the ability of the nanoplasmonic platformto be read using LSPR.

To bind the one or more surface recognition elements such as antibodiesand capture moieties to the gold nanoparticles, a modified supportsurface was formed by preparing a surface of the plurality of goldnanoparticles using a mercaptoundecanoic acid to form carboxyl groups,reacting N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloridewith the carboxyl groups to form an amine reactive intermediate, andstabilizing the amine reactive intermediate by N-hydroxysulfosuccinimideto form the modified support surface. Then, Protein G was used toimmobilize anti-IFN-γ antibody. Additionally, other or additionalantibodies may be present based on the pathogens and infectious agentsto be detected. It is contemplated that multiple pathogens may bedetected on a single nanoplasmonic platform. In some embodiments, theantibody may be a polyclonal antibody. Additionally, in some forms, themodified support surface is linked to at least one of a protein A, aprotein G, a protein A/G, a Streptavidin protein, and a NeutrAvidinprotein which is used to form chemical bonds to immobilize recognitionelements such as the antibody on the modified support surface.

After the biosensing platform was prepared, a broad range ofconcentrations (1 pg/mL to 1 μg/mL) for IFN-γ spiked in PBS samples wasevaluated, and the results of which are presented in FIG. 20A. In theexperiments, 100 μL of IFN-γ spiked sample was loaded into each antibodyimmobilized surface of the nanoplasmonic platform and incubated at 4° C.for an hour. Following each surface modification step, the surfaces wererinsed with 1×PBS three times to remove unbound IFN-γ and chemicalmoieties. In the selected detection range, the highest detected peakshift was observed as 11.6 nm±0.7 nm at 1 μg/mL of IFN-γ spikedconcentration as illustrated in FIG. 20A. The peak shift decreased withdecreasing IFN-γ concentration, and the lowest shift was observed as 8.6nm±0.8 nm at 1 pg/mL of IFN-γ spiked concentration as illustrated inFIG. 20A. Additionally, the repeatability parameter was observed to be88-94% for IFN-γ detection experiments using nanoplasmonic platform asillustrated in FIG. 20B. Here, a reliable, repeatable, feasible,label-free, and fluorescence-free and repeatable technology is presentedthat detects IFN-γ from biologically relevant solutions such as PBS.

Example XIX

This example describes the capture and detection of intact Hepatitis Bvirus using the nanoplasmonic platform technology.

The surface chemistry for intact Hepatitis B virus (HBV) capture anddetection was started with the modification of the polystyrenesubstrates with poly-l-lysine (PLL) (although other layers might be ableto immobilize the nanoparticles on the substrate and not interfere withLSPR detection). PLL provided amine groups to immobilize goldnanoparticles on the substrate. The nanoplasmonic immobilization layerwas then functionalized with thiol groups, although other attachmentsbetween the layer and the substrate or the gold nanoparticles might alsobe employed as long as it did not impair the ability of thenanoplasmonic platform to be read using LSPR.

To bind the one or more surface recognition elements such as antibodiesand capture moieties to the gold nanoparticles, a modified supportsurface was formed by preparing a surface of the plurality of goldnanoparticles using a mercaptoundecanoic acid to form carboxyl groups,reacting N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloridewith the carboxyl groups to form an amine reactive intermediate, andstabilizing the amine reactive intermediate by N-hydroxysulfosuccinimideto form the modified support surface. Then, protein G was used toimmobilize anti-HBV-HBc (core) and anti-HBV-HBs (surface) antibodies.Additionally, other or additional antibodies may be present based on thepathogens and infectious agents to be detected. It is contemplated thatmultiple pathogens may be detected on a single nanoplasmonic platform.In some embodiments, the antibody may be a polyclonal antibody.Additionally, in some forms, the modified support surface is linked toat least one of a protein A, a protein G, a protein A/G, a Streptavidinprotein, and a NeutrAvidin protein which is used to form chemical bondsto immobilize recognition elements such as the antibody on the modifiedsupport surface.

Following the quantification of the stock sample, HBV spiked in culturemedia and PBS samples were prepared for the final concentration rangingfrom 10 to 10⁵ IU/mL. The unit of measurement (i.e., IU/mL) equals to5-6 genome copies/mL. 100 μL of HBV spiked sample was loaded into eachantibody immobilized surface of the nanoplasmonic platform and incubatedat 4° C. for an hour. PBS and media samples without HBV spiking wereused as controls. Following each surface modification step, the surfaceswere rinsed with 1×PBS three times to remove unbound HBV and chemicalmoieties. In HBV-HBc detection experiments, the highest detected peakshift was observed as 8.8±0.6 nm at 10⁵ IU/mL of HBV concentration asillustrated in FIG. 21A. The peak shift decreased with decreasing HBVconcentration, and the lowest shift was observed as 2.5 nm±0.6 nm at 10IU/mL of HBV concentration as illustrated in FIG. 21A. The repeatabilityparameter was observed to be 79-94% for HBV-HBc capture and detectionexperiments using nanoplasmonic platform as illustrated in FIG. 21B. InHBV-HBs detection experiments, the highest detected peak shift wasobserved as 8.9±0.6 nm at 10⁵ IU/mL of HBV concentration as illustratedin FIG. 22A. The peak shift decreased with decreasing HBV concentration,and the lowest shift was observed as 3.0±0.5 nm at 10 IU/mL of HBVconcentration as illustrated in FIG. 22A. The repeatability parameterwas observed to be 82-98% for HBV-HBs capture and detection experimentsusing nanoplasmonic platform as illustrated in FIG. 22B. Thelimit-of-detection was evaluated for HBV-HBc and HBV-HBs experiments bycomparing the control (culture media and PBS samples without HBV) andthe lowest concentration of HBV spiked samples. Here, the statisticalanalysis was performed using one-way analysis of variance (ANOVA) withTukey's posthoc test followed by Bartlett's test for equal variances formultiple comparisons, and statistical significance threshold was set at0.05 (n=6, p<0.05). The statistical analyses demonstrated that the limitof detection was observed to be 10-100 IU/mL in culture media and PBSsamples. Here, we present a reliable, repeatable, feasible, label-free,and fluorescence-free and repeatable technology that captures anddetects intact HBV from biologically relevant solutions such as PBS,blood and culture media.

Many modifications and variations to this preferred embodiment will beapparent to those skilled in the art, which will be within the spiritand scope of the invention. Therefore, the invention should not belimited to the described embodiment. To ascertain the full scope of theinvention, the following claims should be referenced.

What is claimed is:
 1. A nanoplasmonic platform for the detection and quantification of at least one biological moiety in a sample, the nanoplasmonic platform comprising: a substrate having an immobilization layer; a plurality of nanoparticles immobilized on the immobilization layer; and at least one recognition element linked to the nanoparticles, the at least one recognition element configured to selectively bind to the at least one biological moiety; wherein the nanoplasmonic platform is adapted for the detection and quantification of the at least one biological moiety using localized surface plasmon resonance.
 2. The nanoplasmonic platform of claim 1, wherein the plurality of nanoparticles are comprise gold, wherein the at least one biological moiety comprises a pathogen, and wherein the at least one recognition element comprises an antibody.
 3. The nanoplasmonic platform of claim 1, wherein the substrate is optically transparent.
 4. The nanoplasmonic platform of claim 3, wherein the substrate comprises at least one of polystyrene, glass parylene, quartz crystal, graphene and mica layers, and poly(methyl methacrylate).
 5. The nanoplasmonic platform of claim 1, wherein the immobilization layer is poly-l-lysine and has amine-terminated groups that are used to immobilize the plurality of nanoparticles.
 6. The nanoplasmonic platform of claim 1, wherein the immobilization layer is functionalized with at least one of amine group or thiol groups.
 7. The nanoplasmonic platform of claim 1, wherein the at least one recognition element is a polyclonal antibody.
 8. The nanoplasmonic platform of claim 1, wherein the at least one recognition element includes at least one of an anti-gp120 antibody, an anti-gp41 antibody, an anti-gp24 antibody, and lectin and the at least one biological moiety includes HIV.
 9. The nanoplasmonic platform of claim 8, wherein the anti-gp120 antibody is adapted to bind to multiple subtypes of HIV.
 10. The nanoplasmonic platform of claim 8, wherein, to link the at least one recognition element to the nanoparticles, a modified support surface is formed by preparing a surface of the plurality of nanoparticles using a mercaptoundecanoic acid to form carboxyl groups, N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride is reacted with the carboxyl groups to form an amine reactive intermediate, and the amine reactive intermediate is stabilized by N-hydroxysulfosuccinimide to form the modified support surface.
 11. The nanoplasmonic platform of claim 8, wherein the modified support surface is linked to at least one of a protein A, a protein G, a protein AG, a Streptavidin protein, and a NeutrAvidin protein which is used to immobilize the anti-HIV gp120 antibody.
 12. The nanoplasmonic platform of claim 1, wherein, after binding of the biological moiety to the recognition element, the nanoplasmonic platform exhibits an observable change in at least one of wavelength shift and extinction intensity.
 13. The nanoplasmonic platform of claim 1, wherein the at least one biological moiety includes multiple pathogens.
 14. The nanoplasmonic platform of claim 1, wherein the nanoplasmonic platform is based on a microfluidic device having an inlet for reception of the sample in which the inlet is in fluid communication with a capture detection channel that includes at least one recognition element linked to the plurality of nanoparticles.
 15. The nanoplasmonic platform of claim 14, wherein the microfluidic device further comprises a filter disposed between the inlet and the capture detection channel, the filter having a porosity that filters the sample to produce a filtered sample for selectively binding to the at least one recognition element.
 16. A method of making a nanoplasmonic platform comprising: depositing an immobilization layer on a substrate; immobilizing a plurality of nanoparticles on the immobilization layer; and linking at least one recognition element to the nanoparticles, the at least one recognition element configured to selectively bind to at least one biological moiety.
 17. The method of claim 16, wherein the plurality of nanoparticles are comprise gold, wherein the at least one biological moiety comprises a pathogen, and wherein the at least one recognition element comprises an antibody.
 18. The method of claim 16, wherein the substrate is optically transparent.
 19. The method of claim 16, wherein the substrate comprises at least one of polystyrene, glass, parylene, and poly(methyl methacrylate).
 20. The method of claim 16, wherein the at least one recognition element is a polyclonal antibody.
 21. The method of claim 16, wherein the at least one recognition element includes at least one of an anti-gp120 antibody, an anti-gp41 antibody, an anti-gp24 antibody, and lectin and the at least one biological moiety includes HIV.
 22. The method of claim 16, wherein a modified support surface for linking the at least one recognition element to the nanoparticles is formed by the steps of: preparing a surface of the plurality of nanoparticles using a mercaptoundecanoic acid to form carboxyl groups; reacting N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride with the carboxyl groups to form an amine reactive intermediate; and stabilizing the amine reactive intermediate using the of N-hydroxysulfosuccinimide to form the modified support surface.
 23. The method of claim 22, wherein the modified support surface is linked to at least one of a protein A, a protein G, a protein AG, a Streptavidin protein, and a NeutrAvidin protein which is used to immobilize an anti-gp120 antibody.
 24. A method of detecting a biological moiety in a sample using the nanoplasmonic platform of claim 1, the method comprising: receiving the sample on the nanoplasmonic platform to capture the biological moiety; performing localized surface plasmon resonance on the nanoplasmonic platform to obtain at least one of a wavelength shift and an extinction intensity corresponding to the sample received on the nanoplasmonic platform.
 25. The method of claim 24, further comprising the step of correlating the at least one of the wavelength shift and the extension intensity corresponding to the sample received on the nanoplasmonic platform to determine whether the biological moiety has bound to the at least one recognition element on the nanoplasmonic platform.
 26. The method of claim 24, further comprising the step of correlating the at least one of the wavelength shift and the extinction intensity corresponding to the sample received on the nanoplasmonic platform to determine a quantity of the biological moiety present in the sample.
 27. The method of claim 24, wherein a biological moiety is a subtype of HIV and the nanoplasmonic platform is adapted to detect multiple subtypes of HIV.
 28. The method of claim 24, wherein the nanoplasmonic platform is based on a microfluidic device having an inlet for reception of the sample in which the inlet is in fluid communication with a capture detection channel that includes at least one recognition element linked to the plurality of nanoparticles and the step of receiving the sample on the nanoplasmonic platform includes flowing the sample through the capture detection channel to selectively bind the biological moiety to the at least one recognition element.
 29. The method of claim 28, wherein the microfluidic device further comprises a filter disposed between the inlet and the capture detection channel and the method further comprises the step of filtering the sample to produce a filtered sample prior to the step of selectively binding the biological moiety to the at least one recognition element in the capture detection channel. 